Production of Exopolysaccharides by a Submerged Culture of an Entomopathogenic Fungus, Paecilomyces sp.

Abstract

Exopolysaccharide basic was obtained from a submerged culture of a native Paecilomyces sp. strain isolated from Chilean soil.

Full text

Production of Exopolysaccharides by a Submerged Culture of an
Entomopathogenic Fungus, Paecilomyces sp.
Luis Lillo
a,
*, Julio Alarco
´n
a
, Gerardo Cabello
a
, Sergio A
´guila
b
,
and Joel B. Alderete
b
a
Departamento Ciencias Ba
´sicas, Facultad de Ciencias, Universidad del Bı
´o-Bı
´o, Chilla
´n,
Chile. Fax: +56-42-253046. E-mail: llillo@ubiobio.cl
b
Departamento Quı
´mica Orga
´nica, Facultad de Ciencias Quı
´mica, Universidad de
Concepcio
´n, Concepcio
´n, Chile
* Author for correspondence and reprint requests
Z. Naturforsch. 62 c, 576Ð578 (2007); received January 29/March 13, 2007
Exopolysaccharide basic was obtained from a submerged culture of a native Paecilomyces
sp. strain isolated from Chilean soil.
Key words: Entomopathogenic Fungi, Exopolysaccharides, Paecilomyces sp.
Introduction
The microbial exopolysaccharides (EPS) are a
class of high value biopolymers with a wide variety
of industrial applications. Various types of EPS in
the different fields of medicine, foods, cosmetics
and other industries have been used (Kim et al.,
2003). In particular, many kinds of EPS have been
produced from submerged cultures of mushrooms
or entomopathogenic fungi (Xu et al., 2003). An-
other exopolysaccharide, 154Ð2-amino-2-deoxy-
α-d-galactan, also known as poly-α-d-galactosam-
ine, was obtained from the culture fluid of the fun-
gus Paecilomyces sp. I-1 (Takagi and Kadowaki,
1985). Poly-α-d-galactosamine may be constituted
as an important starting material for fine chemi-
cals and biologically active derivatives. It is known
that it exhibits antitumoural effects against solid
tumours transplanted in mice (Lillo and Matsu-
hiro, 2003). It shows similar physicochemical prop-
erties like chitosan, a linear polysaccharide of
2-amino-2-deoxy-d-glucopyranose, which is an
abundant resource available by N-deacetylation of
chitin. Chitosan is industrially produced from crab
shell waste, but research has been carried out on
the use of alternative sources for chitosan (Nieder-
hofer and Müller, 2004).
Paecilomyces is a genus of filamentous fungi
closely related to Penicillium (Brown and Smith,
1957). Taxonomically, the genus is subdivided into
two sections. Section Paecilomyces contains meso-
philic, thermophilic and thermotolerant species
and colonies with yellow-brown to brownish col-
ours. Section Isarioidea contains mesophilic spe-
0939Ð5075/2007/0700Ð0576 $ 06.00 ”2007 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com ·
D
cies with purple-pink, green- or yellow-coloured
colonies (Samson, 1974). However, only EPS pro-
duction of the Paecilomyces section has been re-
ported.
In the present study, we describe the production
of basic EPS obtained in a submerged culture of
Paecilomyces sp.
Experimental
General experimental procedures
FT-IR spectra of KBr pellets were recorded in
the 4000Ð400 cm
Ð1
region using a Shimadzu FT-
IR 8400 instrument. Derivation, including Sa-
vitzky-Golay algorithm with 25 smoothing points,
was performed using the OPUS/IR.
Organism collection
Paecilomyces sp. were cultured in potato dex-
trose agar. Stock cultures were maintained on the
same medium and transferred to fresh medium at
a four weeks interval. A voucher specimen of the
fungus is deposited in the fungi collection of the
Departamento de Ciencias Ba
´sicas, Universidad
del Bı
´o-Bı
´o, Chilla
´n, Chile.
Fungal strain and culture conditions
The native strain of Paecilomyces sp.(PAE-
UBB-001) was grown in shaken-flask culture
Hagen medium containing the following chemicals
(per liter of distilled water): 0.05 g CaCl
2
·2H
2
O
(Merck), 0.025 g KH
2
PO
4
(Merck), 0.25 g

L. Lillo et al. · Exopolysaccharides 577
(NH
4
)
2
HPO
4
(Merck), 0.15 g MgSO
4
·7H
2
O
(Merck), 1.3 ml FeCl
3
1% (Merck ), 3.0 g malt ex-
tract (Merck) and 10 g glucose (Merck). Each
flask containing 100 ml of medium was inoculated
with 2.0 ml suspension of the fungus obtained
from the surface of stock slants.
In a 2,000-ml Erlenmeyer flask containing
500 ml of medium with aeration and agitation (150
rpm), the fermentation was performed. 125 ml of
well grown culture (7 d) in the same medium were
used as inoculum. The fermentation was stopped
after 30 d. The pH value of the medium was ad-
justed to 6.5 with HCl (2 m)orKOH(2m).
Mycelial dry weight and EPS determination
The mycelial dry weight was measured after re-
peated washing (with distilled water) of the myce-
lial pellet, obtained after filtration, and then dry-
ing at room temperature for 12 h. The weight was
compared to the total weight obtained from the
filtrates.
The resulting culture filtrate was mixed with
four volumes of absolute ethanol, stirred vigor-
ously, and kept overnight at Ð10 ∞C. The precipi-
tate was centrifuged at 3,000 rpm for 15 min and
the supernatant was discarded. After repeated
precipitation steps, the resulting EPS were dia-
lyzed at room temperature overnight in de-ionized
water and lyophilized, and the weight of EPS
was estimated.
Results and Discussion
Paecilomyces sp. is an entomopathogenic fungus
and a good alternative to chemical control of
nematods. We isolated this fungus from soil. EPS
were isolated from liquid medium and precipitated
with ethanol. The FT-IR spectrum (Fig. 1A) shows
characteristic absorption bands at 3403.93 cm
Ð1
as-
signed to an N-H and O-H stretching, at
2928.49 cm
Ð1
assigned to a C-H stretching, at
1653.81 cm
Ð1
assigned to an N-H bending and at
1411.29 cm
Ð1
due to a C-O deformation of a sec-
ondary alcoholic group. The broad band centred
at 1653.81 cm
Ð1
is resolved into two bands, in the
second derivative mode (Fig. 1B), one at
1661.13 cm
Ð1
assigned to a C=O stretching vibra-
tion of the N-acetyl group and another at
1527.84 cm
Ð1
assigned to the N-H deformation vi-
bration of a primary amine group (Conley, 1966).
These results suggest that the EPS are polysaccha-
rides composed of galactosamine residues. The
Fig. 1. FT-IR spectrum of EPS isolated from Paecilomy-
ces sp. and second derivative FT-IR spectrum of EPS.
Fig. 2. FT-IR spectrum of chitosan.
EPS present similar characteristic functional
groups like chitosan (Lillo and Matsuhiro, 2003).
The FT-IR spectrum (Fig. 2) of chitosan shows two
characteristic bands; they are present in the FT-IR

578 L. Lillo et al. · Exopolysaccharides
spectrum of EPS at 1654.80 cm
Ð1
assigned to a C=
O stretching vibration of an N-acetyl group and at
1596.94 cm
Ð1
due to an N-H stretching of a pri-
mary amine group (Lillo and Matsuhiro, 1997).
Fig. 3 shows the growth kinetics of Paecilomyces
sp. and the production of EPS. The major produc-
tion of EPS is obtained, four-day culture (0.6379 g
L
Ð1
). On the other hand, the concentration of the
EPS is inversely proportional to the increase in
the biomass of the fungus that may be observed.
This decrease in the production of EPS probably
could be due to the exhaustion of the carbon
source in culture medium. Some authors reported
that an excess of carbohydrate in the growing me-
dium is necessary for stimulating the biosynthesis
of EPS (Kojic et al., 1992; Xu and Yun, 2004; Xu
et al., 2006).
Paecilomyces sp. isolated from soil is a good re-
source for the production of basic polysaccharides.
Nevertheless, it is necessary to continue the study
to find the best conditions for a high EPS produc-
tion. This type of molecules presents important ap-
plications for pharmaceutical purposes due to
their diverse biological activities.
In conclusion, a combination of medium compo-
sition and environmental conditions should be
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Acknowledgement
We are grateful to Direccio
´n de Investigacio
´n
de la Universidad del Bı
´o-Bı
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055609 3/R).
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