Appl Microbiol Biotechnol (2009) 81:827–837
DOI 10.1007/s00253-008-1700-2
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Production and structural analysis of the polysaccharide
secreted by Trametes (Coriolus) versicolor ATCC 200801
Udo Rau & Anja Kuenz & Victor Wray & Manfred Nimtz &
Julika Wrenger & Hasan Cicek
Received: 3 June 2008 / Revised: 29 August 2008 / Accepted: 1 September 2008 / Published online: 18 September 2008
# Springer-Verlag 2008
Abstract Trametes versicolor ATCC 200801 secretes
4.1 g L−1 of exopolysaccharide (EPS) when synthetic
minimal medium and low-shear bioreactor cultivation
technique are used. Structural and compositional analyses
by thin layer chromatography, gas chromatography–mass
spectrometry, electrospray ionization tandem mass spec-
trometry, and nuclear magnetic resonance spectroscopy
yielded predominantly glucose and small amounts of
galactose, mannose, arabinose, and xylose. The main EPS
is composed of β-1,3/β-1,6-linked D-glucose molecules
which is identical with Schizophyllan but does not possess
a triple helical arrangement as secondary structure. Two
molar mass fractions were detected by size exclusion
chromatography yielding weight-average molecular weights
of 4,100 and 2.6 kDa. Protein content varies between 2–
3.6% (w/w). The exopolysaccharide is different in the nature
of the glycosidic linkage, composition of monosaccharides,
protein content, and weight-average molecular weight
U. Rau (*) : J. Wrenger : H. Cicek
Institute of Biochemistry and Biotechnology,
Technical University Braunschweig,
Spielmannstr. 7,
38106 Braunschweig, Germany
e-mail: U.Rau@tu-bs.de
A. Kuenz
Institute of Agricultural Technology and Biosystems Engineering,
Johann Heinrich von Thünen-Institut,
Bundesallee 50,
38116 Braunschweig, Germany
V. Wray : M. Nimtz
Department of Structure Biology,
Helmholtz Centre for Infection Research,
Inhoffenstr. 7,
38124 Braunschweig, Germany
compared to the well-known polysaccharopeptide (PSP)
and polysaccharopeptide Krestin (PSK).
Keywords Trametes versicolor . Polysaccharide .
Bioreactor cultivation . Structure analysis . Viscosity .
Molecular weight
Introduction
Many fungi are able to produce extracellular polysacchar-
ides. They fulfill different tasks during the growth on
natural substrates, such as adhesion to surfaces, immobili-
zation of secreted enzymes, prevention of hyphae from
dehydration and increased residence time of nutrients inside
the mucilage (Rau 1997). Many of them contain α-
(Pullulan) or β-linked (e.g., Scleroglucan, Schizophyllan)
glucose units. The alignment and disposition of linkage and
branching affect the three-dimensional structure and deter-
mine the physicochemical characteristics of the gum. The
branched β-glucans are biologically active and consequently
are used in medicine and biotechnology, as well as additives
in food and cosmetics (Manzoni and Rollini 2001).
During the Ming Dynasty, Li Shi Zhen recorded in the
Compendium of Materia Medica the healing properties of
the mushroom Trametes (Coriolus) versicolor, which
contains protein-bound polysaccharides. More than 120
strains of T. versicolor are known (Ng 1998). The
polysaccharopeptide Krestin (PSK) and polysaccharopep-
tide (PSP) are two chemically related products from T.
versicolor CM-101 and Cov-1, respectively, that have been
characterized (Sakagami et al. 1991). PSK is recovered
from hot water extracts of the biomass by salting out with
ammonium sulphate, whereas PSP is isolated by alcoholic
precipitation from a hot water extract. Likewise, ultrasonic
828
extraction is applicable for downstream processing (Yu-
Wen et al. 2006). These polymers are classified as
biological response modifiers and are used in conjunction
with cancer therapy and as immunoenhancers (Cui and
Chisti 2003).
During an investigation related to the laccase activity of
T. versicolor ATCC 200801 (Taspinar and Kolankaya
1998), Aktas et al. (2001) noticed that a hitherto unknown
exopolysaccharide was secreted only if the complex
cultivation medium was changed to synthetic minimal
medium. Here, we present details of the characterization
of the polymer secreted by this strain including its
production profile in shake flasks and bioreactors, as well
as an elucidation of its structure for the first time.
Materials and methods
Microorganism Trametes versicolor ATCC 200801 was
isolated and deposited by O. Yesilada, Adana, Turkey
(Yesilada et al. 1998). Stock cultures of the strain were
grown for 7 days at 30 °C on potato dextrose agar (39 g L−1)
supplied with 5 g L−1 yeast extract. They were stored at
4 °C and subcultured every month.
Media composition and culture conditions For precultiva-
tion, 5 mL sterilized water was added into one well-grown
agar tube. After thorough mixing, this suspension was
poured into 500-mL medium contained in 2,000-mL
Erlenmeyer flasks without baffles. The medium for shake
flask and bioreactor cultivation contained 20 g L−1 glucose,
20 mL L−1 salt stock solution, and 1 mg L−1 thiamine
(sterile filtrated). The salt stock solution was composed of
15 g sodium citrate · 5H2O, 25 g KH2PO4, 10 g NH4NO3,
1 g MgSO4 · 7H2O, 0.5 g CaCl2 · 2H2O, and 1-mL trace
element solution dissolved in 100 mL deionized water.
CaCl2 · H2O was separately dissolved in 10 mL deionized
water and added to the stock salt solution with stirring.
The trace element solution contained 0.5 g citric acid · H2O,
0.5 g ZnSO4 · 7H2O, 0.1 g Fe(NH4)2(SO4)2 · 6H2O, 25 mg
CuSO4 · 5H2O, 5 mg MnSO4 · H2O, and 5 mg H3BO3 and
5 mg H3(PMo8O40) · H2O dissolved in 20 mL deionized
water. This preculture was incubated on a rotary shaker at
150 rpm, 30 °C and pH 4.7 (not controlled) for 5 days. The
homogenized (60 s, Ultra Turrax T 25, Janke & Kunkel,
Staufen, Germany) culture suspension was used as 3% (v/v)
inoculum for the second precultivation which was incubat-
ed under the same conditions as above.
A 42-L bioreactor (Sartorius BBI Systems, Melsungen,
Germany) filled with 30-L medium and equipped with three
four-bladed fan impellers was used. The cultivation
conditions were 30 °C, pH 4.7 (not controlled), 60–
70 rpm, and aeration rate 180–720 L h−1. Glucose was
Appl Microbiol Biotechnol (2009) 81:827–837
separately sterilized and thiamine sterile filtered. Both
components were added to the medium aseptically. Three
liters of the homogenized second preculture was used as
inoculum. All experiments were carried out at least in
duplicate to ensure reproducibility.
Cultivation parameters Biomass was determined by appro-
priate dilution of the culture suspension with deionized
water, subsequent homogenization (Ultra Turrax), and
centrifugation at 15,000×g. The pellet was washed twice
with deionized water and dried to constant weight at 80 °C
for 48 h. The cell-free supernatant was kept at 4 °C for
further analysis. For exopolysaccharide (EPS) determina-
tion, two volumes of 2-propanol were added to the
supernatant, which was subsequently stored at 4 °C for
1 h to complete precipitation. After centrifugation (10 min,
15,000×g), the gum was dried to constant weight at
100 mbar and 40 °C for 48 h. Glucose content was deter-
mined with an Accutrend™ glucose analyzer (Boehringer
Mannheim, Germany) and the protein content of aqueous
polysaccharide solutions was detected by the Lowry
method (Lowry et al. 1951). Carbon dioxide evolution rate
was calculated online (FERMVis, BlueSens gas sensor
GmbH, Herten, Germany) by gas phase balance and
detection of %CO2 and %O2 (BCpreFerm, BlueSens gas
sensor GmbH, Herten, Germany) in the exhaust.
NMR One- (1H) and two-dimensional (1D/2D; correlation
spectroscopy (COSY), 1H-detected 13C–1H heteronuclear
multiple bond correlation) nuclear magnetic resonance
(NMR) spectra of the compounds were recorded either on
AMX-300 (Bruker, 300 MHz) or AVANCE DMX-600
(Bruker, 600 MHz) spectrometers at 300°K locked to the
deuterium resonance of the solvent D2O. One-dimensional
13
C NMR spectra were recorded at 353°K with dimethyl
sulfoxide-d6 as solvent. Chemical shifts were referenced to
the appropriate solvent signals.
GC-MS and ESI-MS In order to analyze the sugar
compounds, the monosaccharides were detected by gas
chromatography–mass spectrometry (GC–MS) as the
corresponding methyl glycosides after methanolysis and
trimethylsilylation (Chaplin 1982). For the determination of
the linkage positions between monosaccharide residues
using methylation analysis, the freeze-dried polysaccharide
was permethylated (Anumula and Taylor 1992), then
hydrolyzed (4 N trifluoroacetic acid (TFA), 2 h, 100 °C),
reduced with NaBH4, peracetylated, and the resulting
partially methylated alditol acetates analyzed by GC-MS.
For electrospray ionization tandem mass spectrometry (ESI-
MS), the partially hydrolyzed polysaccharide (0.5 N TFA,
2 h, 100 °C) was reduced and permethylated. The
oligosaccharides (3 μL dissolved in 50 μM NaCl solution
Appl Microbiol Biotechnol (2009) 81:827–837
of MeOH (methanol)/water 9:1) were placed into a gold-
coated nanospray glass capillary (Protana, Odense, Den-
mark). The tip of the capillary was located orthogonally in
front of the entrance of a quadrupole time-of-flight (TOF)
mass spectrometer (Micromass, Manchester, UK) equipped
with a nanospray ion source. A voltage of ∼1,000 V was
applied. For collision-induced dissociation experiments,
parent ions were selectively transmitted from the quadru-
pole mass analyzer into the collision cell. Argon was used
as the collision gas and the kinetic energy was set to
∼65 eV. An orthogonal TOF mass analyzer separated the
resulting daughter ions (Nimtz et al. 1996, 1997).
IR Tensor 27 (Bruker Optics, Germany) Fourier transfor-
mation infrared (IR) spectrometer was used in combination
with diamond attenuated total reflectance measurement
using infrared transmitting fibers.
Shear viscosity Low-shear rotational viscometer (RV 100,
Haake, Germany) equipped with sensor system DA 45
(combination of Couette and Searle measurement type) was
used for the determination of shear viscosity.
Size exclusion chromatography The molecular weight of
the isolated EPS was analyzed by size exclusion chroma-
tography (SEC; LaChroma L-7series, Merck-Hitachi,
Tokyo, Japan) using a Suprema column (length 300 mm,
particle size 10 μm; PSS GmbH, Mainz, Germany) with a
porosity of 0.3 μm (resolution 1–30,000 kDa) and a
refractive index detector (Shodex RI (refractive index)
101, Showa Denko Europe GmbH, München, Germany).
Five hundred microliters of 2 g L−1 EPS in dimethyl
sulfoxide (DMSO) was injected. The elution was carried
out at 40 °C with an aqueous solution of 0.2 g L−1 NaN3 at
a flow rate of 0.5 ml min−1. The calibration was performed
by using various Pullulan fractions (PSS GmbH, Mainz,
Germany) with molecular weights of 1.32, 5.9, 11.8, 22.8,
47.3, 112, 212, 404, 788, and 1,660 kDa.
Hydrolysis with sulfuric acid Two grams of the precipitated
polysaccharide was dissolved in 10 mL 2 M H2SO4 and
heated at 121 °C and one bar for 1 h. The hydrolyzate was
diluted 1:2 (v/v) with distilled water and neutralized with
BaCO3. The precipitated BaSO4 was removed by centrifu-
gation (5 min; 10,000×g). Remaining solids were separated
by membrane filtration (SM 13400, Sartorius, Göttingen,
Germany). The distinct separation of single spots for
qualitative sugar analyses by thin layer chromatography
(TLC) was carried out by the development on Silica gel 60
F254 (Merck, Germany) plates with the ternary solvent
system CHCl3/MeOH/H2O (65:15:2, v/v/v). After drying,
the sugar spots were visualized by spraying anisic alde-
hyde/sulfuric acid/glacial acetic acid (0.5:1:50, v/v/v) or α-
829
naphthol/sulfuric acid/ethanol/water (17:10.5:65.5:6.5, v/v/
v/v) with subsequent heating of the plates. The quantitative
isolation of the different sugar components was performed
on a LiChroprep silica gel 60 column (Merck, Germany)
using different solvent mixtures of CHCl3/MeOH/H2O
(65:15:2, 65:35:4, 65:50:7, v/v/v). The sugar containing
fractions were identified by TLC and analyzed by NMR
spectroscopy and GC-MS.
Hydrolysis with trifluoroacetic acid The polysaccharide
was treated with 0.05, 0.1, 0.5, 0.75, 1.0, and 2.0 M TFA
at 100 °C for 2 or 5 h as follows: (1) 30 mg freeze-dried
polysaccharide was dissolved in 3 mL 0.1, 0.5, 0,75, 1.0, or
2.0 M TFA and heated for 5 h at 100 °C. The sample was
freeze-dried again. (2) Thirty-milligram freeze-dried poly-
saccharide was dissolved in 3 mL 0.05 M TFA for 2 h at
100 °C. This solution was added to three volumes of 2-
propanol. The precipitate was separated by filtration. The
filtrate was evaporated, washed twice with distilled water,
and freeze-dried. The precipitate was dissolved in distilled
water, precipitated again twice with 2-propanol, and finally
freeze-dried.
Hydrolysis with β-glucanase The enzymatic cleavage was
performed with a commercially available β-glucanase
(CelluPract AL100, Biopract, Berlin, Germany) isolated
from Trichoderma reesei M18.2. 50 mL aqueous polysac-
charide solution (5 g L−1, pH 4.7) was incubated with
125 μL enzyme at 50 °C for 90 min. The reaction was
stopped using an ice bath and monitored by TLC. The
resulting fractions were quantitatively separated on a
LiChroprep silica gel 60 column and analysed by GC–MS
and NMR spectroscopy.
Application of protease One-gram polysaccharide, precipi-
tated by 2-propanol, was dissolved in 10 mL phosphate
buffer (pH 7.8). One-milliliter protease suspension (0.01 g
subtilisin Carlsberg (EC 3.4.21.62), Novozyme, Denmark
in 10 mL phosphate buffer pH 7.8) was added and
incubated at 37 °C for 1 h. Enzyme activity was terminated
by boiling the suspension. After annealing, 2 mL of
saturated NaCl solution and 4 mL sulfosalicylic acid were
added to precipitate unhydrolyzed protein. After centrifu-
gation and filtration (membrane filter SM 13400, Sartorius,
Germany) of the supernatant, the polysaccharide was
precipitated with 2-propranol, redissolved in water, and
the protein content determined.
Results
Cultivation The cultivation of T. versicolor in shake flasks
without baffles yielded maximum biodry mass and exopo-
830 Appl Microbiol Biotechnol (2009) 81:827–837

lysaccharide of 3.8 and 3.9 g L−1, respectively, after
10 days. Growth and exopolysaccharide formation were
diminished if flasks with two baffles were used (data not
shown). This result indicated a distinct shear sensitivity of
the fungus. For this reason, low-shear fan impellers were
installed for bioreactor cultivation (Fig. 1).
Cultivation of T. versicolor in bioreactor showed that the
pO2 dropped to zero after 3.5 days and caused oxygen
limitation for the first time. An increase of the aeration rate
from 180 to 360 and 720 L h−1 at day 3.9 and 4.9,
respectively, only led to a transient reduction of oxygen
limitation. Increasing the impeller speed from 60 to 70 rpm
resulted in a similar effect on the pO2-signal as the first
increase of aeration rate. However, foam formation was
enhanced; hence, the stirrer speed was subsequently
reduced to the initial value which implicates permanent
oxygen limited conditions. Besides small amounts of
hyphal fragments and mycelia, the fungus grew primarily
in pellet form with about 2 mm in diameter (Fig. 1).
Excess polysaccharide is secreted over the cell wall into
the supernatant. However, the polysaccharide is also part of
the cell wall (Sietsma et al. 1985) and essential for growth.

Fig. 1 Thirty-liter bioreactor

cultivation of T. versicolor Stirrer speed (rpm) = 60 =70 =60
equipped with three four-bladed
fan impellers at 30 °C with Aeration rate (L h-1)=180 =360 =720
5 20
glucose as carbon source. pH
was not controlled. Variation of
aeration rate and/or stirrer speed
occurred at day 3.9, 4.9, 6,
and 7, respectively. Insert: 4
morphology of the pellets. pO2 = 15
oxygen partial pressure of liquid
phase, BM=biomass, EPS=

Glucose (g L )
-1
exopolysaccharide 3 BM
EPS
Glucose 10

5
1

0 0
100 6

pO2
pH
80

1 cm 5
60
pO2 (%)

40
pH

3
2

0 2
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (days)
Appl Microbiol Biotechnol (2009) 81:827–837
Therefore, the polysaccharide represents a primary metab-
olite and formation only occurs during growth of the
fungus. Figure 1 shows that the EPS formation started
coupled with growth and entered the maximum formation
rate of 0.044 g L −1 h−1 during the oxygen limited phase
after 6.3 days. After 7.9 days, the maximum specific
growth rate of 0.02 h−1 was attained. Glucose as energy
and carbon source was totally consumed after 11 days. At
the end of cultivation, 3.7 g L−1 biodry mass and 4.1 g L−1
exopolysaccharide were attained. From these data, the yield
coefficients 0.18 (biomass/glucose), 0.20 (EPS/glucose),
and 1.09 (EPS/biomass) can be calculated. Integration of
the CO2-formation rate (g L−1 h−1; data not shown) yielded
the evolution of 12.4 g L−1 CO2 after 12 days. Application
of the carbon balance with 45% (w/w) and 44% (w/w)
carbon contained in biomass and EPS, respectively, resulted
in 85% recovery related to 20 g L−1 glucose consumed. The
15% of carbon not assigned could be attributed to the
formation of organic acids which decreased the uncon-
trolled pH from 4.7 to 3.4.
After appropriate dilution with water and centrifugation
of the cells, the EPS was isolated by precipitation with 2-
propanol and subsequently lyophilized. Resolvation in
water was possible by heating to 60 °C accompanied with
vigorous stirring. Depending on the time of sampling
during cultivation, 1 g L−1 aqueous redissolved polysac-
charide solution contained only 2–3.6% (w/w) of protein
equivalent to 20–36 mg L−1. After application of the
protease subtilisin, proteins were no longer detected.
Component analysis of the exopolysaccharide The IR-
spectrum showed characteristic peaks at 889 and
814 cm−1. The first adsorption suggests a β-glycosidic
linkage. Normally, the α-glycosidic linkage is indicated by
an adsorption at 840 cm−1. The shift of the second peak to a
shorter wavelength probably suggests only small contin-
gents of α-glycosidic linkages are present.
The polysaccharide was first hydrolyzed with sulfuric
acid. TLC of the hydrolyzate showed four distinct spots
A–D (Fig. 2, lane 3). Only glucose could be unambiguously
assigned to spot A by comparison with reference sugars
(glucose, fructose, mannose, sorbose, arabinose, xylose,
rhamnose, ribose). The chromatographic fractions of the
hydrolyzate were also visualized by TLC (Fig. 2, lane 4–8).
Spots A, B, and D were isolated in larger quantities by
preparative TLC. The compounds related to the fractions in
lanes 5 and 6 were further purified by changing the solvent
mixture (CHCl3/MeOH/H2O) from 65:35:4 to 65:15:2 (v/v/v;
Fig. 2, lane 1–2). These fractions corresponding to the pure
spots A–D were subsequently analyzed by NMR spectros-
copy and GC–MS.
An anomeric mixture of α- and β-glucopyranose was
readily identified as the major components for spot A and C
831
Fig. 2 TLC of sugar containing fractions after polysaccharide
hydrolysis with sulfuric acid. Quantitative separation was performed
on a LiChroprep silica gel 60 column. Right—solvent mixture CHCl3/
MeOH/H2O (65:35:4, v/v/v). Lanes 4–8—fractions after chromato-
graphic separation; lane 9—glucose. Left—solvent mixture CHCl3/
MeOH/H2O (65:15:2, v/v/v). Lanes 1–2 reseparation of lanes 5 and 6;
lane 3—hydrolyzate before chromatographic separation. A–D spot
designation
from the cross peaks in the 2D COSY spectra and their
characteristic 1H NMR chemical shifts. Their identity was
confirmed from the GC–MS analysis. Spot B was a more
complex mixture of four monosaccharides whose spin
systems were readily identified from the 2D COSY
spectrum. Careful inspection of this and comparison with
literature data (Bock et al. 1983) indicated that these were
the α- and β-anomers of xylose and arabinose in their
respective pyranose forms. Again, GC–MS confirmed these
assignments. A sugar signal was not detected for spot D by
NMR spectroscopy. GC–MS analysis showed small
amounts of sugars that were probably impurities. The
presence of glucose, galactose, mannose, xylose, and
arabinose were also detected in the native polysaccharide
(Fig. 3) both before and after protease treatment.
Mass spectrometric characterization of the polysaccharide
Detection by TLC showed that hydrolysis with 2–0.75 M
TFA yielded only monosaccharides. The polysaccharide
fragments obtained by 0.5 M TFA treatment were reduced
with NaBD4 and subsequent permethylated for ESI-MS
analysis. The spectrum yielded molecular ions [M+Na]+ at
m/z 698.3 (Hex2Hexitol-1D), m/z 902.4 (Hex3Hexitol-1D),
m/z 1,106.5 (Hex4Hexitol-1D), and m/z 1,310.6 (Hex5
Hexitol-1D). In spite of threefold repetition of the hydro-
lysis with 0.5 M TFA, no evidence for the presence of
pentose residues, easily detectable by a mass shift of
−44 m.u. compared to an additional hexose residue, was
832
Xyl
Ara
Fig. 3 GC–MS spectrum of the native polysaccharide formed by T. versicolor
detected. Additional structural information was obtained
from collision-induced dissociation of oligosaccharide
parent ions. The daughter ion spectrum from 902.4 m/z
showed an inner-ring fragment at m/z 329.1 (3,5A2 accord-
ing to the nomenclature of Domon and Costello 1988),
suggesting the presence of at least one 1,4- or 1,6-linkage
between the hexose units. The complete set of linkage types
present in the polysaccharide was determined by methyla-
tion analysis. Only partially methylated glucitol acetates
were detected corresponding to terminal, 3- and 3,6-
disubstituted glucose in a ratio of approximately 0.3:2:1.
Hydrolysis with 0.1 or 0.05 M TFA yielded glucose, a
polysaccharide that only contains glucose and the mono-
saccharides xylose and mannose.
Enzymatic cleavage of the polysaccharide Further structur-
al information was afforded by enzymatic cleavage with β-
glucanase CelluPract AL100 (BIOPRACT, Germany) and
resulted in the data shown in Fig. 4. The pure β-glucanase
ß-Glucanase
Glucose
Polysaccharide
+ ß-Glucanase Maltose
Hydrolyzate Purified
hydrolyzate
Glucose
Glucose
Gentiobiose
Gentiobiose
Fig. 4 TLC of the polysaccharide hydrolyzate yielded by enzymatic
treatment (β-glucanase AL100, Biopract, Berlin, GER). Pure glucose
and maltose served as reference. Quantitative purification/separation
of the hydrolyzate was performed on a LiChroprep silica gel 60
column. Solvent mixture CHCl3/MeOH/H2O (65:50:7, v/v/v)
Appl Microbiol Biotechnol (2009) 81:827–837
Glc
Man
Gal
solution contained small amounts of glucose. After enzy-
matic cleavage, glucose and a saccharide with higher
molecular weight could be detected in the crude and
purified hydrolyzate. The undegraded polysaccharides were
separated by precipitation. The remaining saccharides were
purified and isolated by chromatography on the LiChropep
silica gel 60 column. The MS data of the unknown
saccharide were compatible with two hexose units. The
high-field (600 MHz) 1D and 2D COSY spectra were
dominated by the signals from one major disaccharide
component and indicated that other components were less
than 10%. The chemical shift and coupling constant data
suggested the presence of α- and β-anomers of gentiobiose
(ratio 44:56). The β-1,6-linkages of the respective gluco-
pyranose units were confirmed from the observation of
cross peaks in the 1H-detected 13C–1H heteronuclear
multiple bond correlation that affords correlations between
13
C- and 1H-nuclei separated by two and three bonds. Thus,
correlations were observed between H-1′ (′ refers to the
nonreducing sugar of the disaccharide) of both anomers
to C-6 and the reverse correlation of H-6 of both anomers
to C-1′.
Structure investigation by NMR spectroscopy 1D and
DEPT-135 13C-NMR spectra of the polysaccharide were
recorded after its precipitation with 2-propanol from the
cell-free supernatant, freeze drying, and resolvation in
deuterated DMSO with ultrasonication at 80 °C. The
spectra were very similar to those found previously for
the homoglucan Schizophyllan produced by the wood
rotting basidiomycete Schizophyllum commune (Muenzberg
et al. 1995) and showed the following chemical shifts: 13C
NMR (DMSO-d6, 39.5 ppm, 353°K) δ=60.6, 60.7, 60.9
(CH2 ×3, C6 free, A/C/D); 68.1 (CH2, C6 linked, B); 68.4,
68.2 (CH ×3, C4, A/B/C); 70.1 (CH, C4, D); 72.3, 72.5,
73.3 (CH ×4, C-2, A/B/C/D); 74.6, 75.9, 76.1, 76.2, 76.3
Appl Microbiol Biotechnol (2009) 81:827–837
(CH ×5, C3, D, C-5, A/B/C/D); 85.7, 86.0, 86.2 (CH ×3,
C3 linked, A/B/C); 102.6, 102.7, 102.8 (CH ×4, C1). The
presence of a well-resolved spectrum indicated a homo-
polysaccharide with a regular repeating unit consisting of
four glucose units from the integrated spectrum and
detection of four C6 methylene carbons. Three equally
intense signals at 86 ppm were only compatible with three
chemically different β-glucopyranosyl moieties with 1,3-
glycosidic linkages, while the low field shift of one of the
C6 signals (68.1 ppm) indicated a branching to a further β-
glucose moiety attached to C6 of one of the 1,3-units.
Consequently, the data are only compatible with the major
polysaccharide consisting of a tetrasaccharide subunit (Glu-
Glu(-Glu)-Glu)x containing only β-glucopyranosyl moieties
(Fig. 5). Signals for pentoses were not detected.
Shear viscosity and molecular weight The cell-free poly-
saccharide solution (4.1 g L−1) of T. versicolor showed
characteristic shear thinning behavior, i.e., decreasing
viscosity with increasing shear rate. At the upper New-
tonian region, zero shear viscosity approached 3,000 mPas
at a shear rate <0.3 s−1. The Ostwald–de Waele power law
(shear viscosity=K*(shear rate)n−1, K=consistency index,
n=flow behavior index) indicated a flow behavior index of
0.22, as calculated from the slope of the data points at
Fig. 6. A shear viscosity of 86.7 mPas was detected at
30 s−1. Changing the solvent from water to DMSO resulted
in a slight reduction of the shear viscosity to 71 mPas at the
same shear rate and polysaccharide concentration.
Due to reduced solubility of the EPS in water after
drying, DMSO was used as solvent for size exclusion
chromatography. The SEC-RI chromatogram showed two
prominent signals (data not shown). A broad one with a
corresponding high polydispersity of 47.4 resulting a
weight-average molecular weight of 4,100 kDa. From the
narrow signal, polydispersity and weight-average molecular
weight of 1.3 and 2.6 kDa could be calculated, respectively.
The large molecular weight polymer possessed a mass
Fig. 5 Primary molecular structure of the polysaccharide secreted by
T. versicolor based on NMR spectra
833
10000
Shear viscosity [mPas]
1000
100
10
-1 0 1 2
10 10 10 10
-1
Shear rate [s ]
Fig. 6 Shear viscosity of the cell-free aqueous polysaccharide
solution (4.1 g L−1) at 20 °C
proportion of 91% (w/w) compared to 9% (w/w) of the
2.6 kDa component.
Discussion
Cultivations performed in shake flasks with baffles showed
essentially decreased growth and polysaccharide produc-
tion. As known for growing filamentous fungi, shear stress
may cause this effect (Rau 2005). In order to minimize
shear stress during bioreactor cultivation, four-bladed fan
impellers with good mixing characteristics (Gura and Rau
1993) for high viscous culture suspensions were used at
low stirrer speeds.
Figure 1 shows that a temporary avoidance of oxygen
limitation is possible by stepwise increase of aeration rate
and stirrer speed. However, the lower stirrer speed was
restored after 1 day to avoid problems caused by foam
formation. The enhanced polysaccharide production, which
is coupled with growth, took place during the oxygen-
limiting phase. This characteristic is known for the
formation of Scleroglucan and Schizophyllan by the fungi
Sclerotium rolfsii and S. commune (Rau and Brandt 1994;
Schilling et al. 1999), respectively. Oxygen limited con-
ditions lead to increased polysaccharide yields but the
glucan synthase is not influenced by oxygen. It is presumed
that the enzyme produces the polymer constantly; however,
reduced oxygen tension causes reduced growth and chitin
formation. Inside the cell wall, the polysaccharide is
covalently linked (R-glucan) over lysine bridges to chitin
(Bartnicki-Garcia 1999). If reduced chitin is available for
linking, higher amounts of polysaccharides can be secreted
because the fungus synthesizes it permanently until the
carbon or nitrogen source is depleted (Rau 2005). Overall,
834
variable oxygen transfer rates did not influence the process
significantly. After 12 days, final biomass of 3.75 g L−1 and
extracellular polysaccharide of 4.1 g L−1 were attained.
Using complex media compounds (potato dextrose broth
and peptone) in an agitated submerged fermentation, Cui
and Chisti (2003) obtained 4 g L−1 representing a total of
extracellular and intracellular T. versicolor polysaccharo-
peptides by hot water extraction of 18 g L−1 mycelia. The
cultivation took 7 days. Separate yields for extra- and
intracellular polysaccharopeptides were not provided. Only
in a later publication dealing with the same microorganism,
however, using milk permeate as substrate, the same
authors differentiated between 1.1 and 0.09 g L−1 of extra-
and intracellular polysaccharopeptides, respectively, yielded
from about 8.9–10.6 g L−1 biomass, also after 7 days of
fermentation (Cui et al. 2007).
Kim et al. (2002) studied the mycelial growth and
exobiopolymer production of various edible mushrooms
during submerged culture in a 5-L bioreactor using different
complex media. After 15 days of cultivation of T. versi-
color, they obtained 1 g L−1 exopolysaccharide as maxi-
mum and 3.6 g L−1 biomass. However, a specific strain
number was not provided. Likewise, Tavares et al. (2005)
cultivated T. versicolor and obtained only 0.7 g L−1
exopolysaccharide in spite of medium optimization. During
the screening of basidiomycetes in shake flask for the
production of exopolysaccharides and biomass, 1.5 and 2.3
EPS from T. versicolor CCB 202 were attained after 7 and
14 days, respectively, with glucose as primary carbon
source (Maziero et al. 1999). Very recently, the T. versi-
color EPS formation in a 20-L bioreactor could be
increased from 0.61 to 1.66 g L−1 by addition of the herbal
extract of Lycium barbarum (Lin et al. 2008). The
cultivation was terminated after 7 days. Table 1 summarizes
Table 1 Bioreactor production of biomass and extracellular polysaccharide by different strains of T. versicolor
Strain Carbon source (g L−1) EPS
(g L−1)
T. versicolor Glucose (20) 4.1
ATCC 200801
T. versicolor LH1 Glucose(40)/Lycium barbarum 1.66
extract (5)
T. versicolor Glucose (100)/yeast extract 1.1
Wr-74 (30)/milk permeate
(2.5 L/7 L total volume)
T. versicolorc Yeast malt extract (20)d 0.7
CS Carbon source, BM Biomass, EPS Extracellular polysaccharide
a
Related to 22 g L−1 reducing sugar consumed
b
Related to 13 g L−1 reducing sugar consumed
c
Without strain number
d
Glucose 10 g L−1 , malt extract 3 g L−1 , peptone 5 g L−1 , yeast extract 2 g L−1
Appl Microbiol Biotechnol (2009) 81:827–837
the recent data obtained by bioreactor cultivation using
T. versicolor.
As known for many cultivations with fungi, an uncon-
trolled pH was used which decreases slowly from 4.7 to 3.4
due to the formation of unknown organic acids. For
comparison, during cultivation of S. rolfsii, the pH
decreases from 4.5 to 2 due to the formation of oxalic acid
(Schilling et al. 2000) and S. commune is able to produce
huge amounts of L-malic acid (Kawagoe et al. 1997).
The extracellular polysaccharide could be isolated by
precipitation with solvents such as 2-propanol, ethanol, or
methanol. This is an easy and fast process. However, after
lyophilization, it is difficult to redissolve the material in
water or DMSO; vigorous stirring for several hours at 60–
70 °C is necessary. This problem is also apparent using the
polysaccharides Scleroglucan and Schizophyllan (Rau
1999).
Aqueous solutions of previously precipitated and lyoph-
ilized polysaccharide contained about 3% (w/w) protein.
After hydrolysis with subtilisin, protein was not detectable
anymore. This characteristic is an essential difference to
PSP and PSK produced by T. versicolor Cov-1 and CM-
101, respectively (Table 2). Both polysaccharopeptides
contain approximately 10–35% (w/w) protein (Cui and
Chisti 2003). Furthermore, the polysaccharopeptides resist
enzymatic proteolysis (Hotta et al. 1981).
Component analysis was performed for structural deter-
mination of the exopolysaccharide. After complete hydro-
lysis with sulfuric acid (Figs. 2 and 3), it was possible to
identify the main component glucose as well as galactose,
mannose, xylose, and arabinose by the use of NMR
spectroscopy and GC–MS. Rhamnose and fucose, which
are contained in PSP and PSK (Cheng et al. 1998; Ng
1998), respectively, were not detected (Table 2).
Biomass YEPS/CS YEPS/BM EPS-productivity References
(g L−1) (g g−1) (g g−1) (g L−1 day−1)
3.75 0.21 1.09 0.34 This study
3.71 0.04 0.45 0.23 Lin et al. 2008
8.9 0.05a 0.12 0.15 Cui et al. 2007
3.8 0.03b 0.18 0.07 Tavares et al. 2005
Appl Microbiol Biotechnol (2009) 81:827–837
Table 2 Molecular composition of the polysaccharides formed by T. versicolor
Strain ATCC 20080 Cov-1
Reference This study Cheng et al. 1998
Polysaccharide This study PSP
Glucose + +
Galactose + −
Mannose + −
Arabinose + +
Xylose + −
Rhamnose − +
Fucose − −
Protein (% w/w) 2–3.6 10
e-PSP Extracellular polysaccharopeptide, + Present, − Not present
For compositional analysis of the polysaccharide, partial
hydrolysis was performed using TFA and a β-glucanase.
After hydrolysis with 0.5 M TFA, 1,3- and 1,6-hexoses of
various chain lengths could be verified. Pentose units as
well as oligosaccharides with side chains were not detected.
The results of hydrolysis with 0.05 M TFA revealed that
glucose is the sole component of the remaining polysac-
charide, while xylose, mannose, and glucose were detected
as monosaccharides. The application of a β-glucanase
yielded glucose as monosaccharide and gentiobiose as
disaccharide.
Careful interpretation of the 1D 13C-NMR data of the
polysaccharide resolved in DMSO-d6 indicated only glu-
cose as monomer component and excluded the presence of
other sugars. However, the achieved NMR pattern is known
(Muenzberg et al. 1995) and is identical with that for
Schizophyllan. The repeating unit is composed of four
glucose units containing the β-1,3-linked main chain with
one β-1,6-linked glucose molecule at every third glucose of
the main chain (Fig. 5). This result agrees with the sole
release of glucose and gentiobiose by the attack of the β-
glucanase. Obviously, the other monosaccharides are
weakly linked/associated to the polymer and were released
by the sample preparation for 13C-NMR. Additionally, they
were detected only in minor concentrations. Thus, glucose
is present as dominant sugar which could imply that the
exopolysaccharide is mainly a glucan and resembles the
polysaccharides formed by T. versicolor Wr-74 and ATCC
20545 (Cui et al. 2007) as well as T. versicolor LH1 (Lin et
al. 2008) which contain also minor amounts of galactose,
mannose, and xylose (Table 2). An identical monosaccha-
ride composition is inexistent compared to the EPS
investigated in this study because arabinose is present.
PSP and PSK consist mainly of α-1,4 and β-1,3 linkages
(Ng 1998). Analysis by GC–MS showed a dominance of
1,4-, 1,2-, and 1,3-glucose linkages together with small
amounts of 1,3-, 1,4-, 1,6-galactose-, 1,3-, 1,6-mannose as
835
CM-101 ATCC 20545 Wr 74 LH 1
Ng 1998 Cui et al. 2007 Lin et al. 2008
PSK EPS e-PSP
+ + +
+ + +
+ + +
− − −
+ + +
− + −
+ − −
28–35 1.7–4.1 13.8–22.6
well as 1,3- and 1,4-arabinose linkages. A study on the
chemical structure of polysaccharide from the fruiting body
of T. versicolor showed that glucose is contained substan-
tially as primary monosaccharide (Zhang et al. 2001).
Methylation analysis combined with NMR indicated β-1,3-
and β-1,4-linked glucose units as main chain with branches
in β-1,3,6 and β-1,4,6-positions. T. unicolor polysaccharide
also contains basically glucose but glycosidic linkages
similar to PSK and PSP (Teng et al. 2007). In contrast, the
polysaccharide investigated here is composed of only β-
1,3- or β-1,6-linked glucose units. Furthermore, the
detected monosaccharides are not identical with those
contained either in PSK or PSP (Table 2).
Tavares et al. (2005) measured the shear viscosity
(maximum 2,000 mPas at <0.1 s−1) of the whole culture
suspension of T. versicolor including 3.8 g L−1 mycelia and
0.7 g L−1 exopolysaccharide. Due to the low polysaccharide
content, it contributes only marginally to the viscosity
yield. The shear viscosity of the cell-free polysaccharide
was not measured. However, the average molecular weight
distribution was determined by SEC to be 67 kDa, which is
comparable with the data for PSK and PSP of approxi-
mately 100 kDa (Cui and Chisti 2003).
The investigated EPS yielded zero shear viscosity of
approximately 3,000 mPas by employing 4.1 g L−1 cell-free
aqueous solution. This result is in accordance with
measurements of 2 g L−1 aqueous polysaccharide solutions
produced by S. rolfsii (Rau and Wagner 1987) which
attained zero shear viscosity of 4,000 mPas at a molar mass
of approximately 10,000 kDa (Stephan 1986). These
findings gave a first indication that the EPS possessed a
higher molar mass than PSK and PSP which were
supported by the results of SEC. Two fractions were
detected with weight-average molecular weights of 4,100
and 2.6 kDa. Furthermore, DMSO as solvent only
marginally reduced the shear viscosity of the EPS com-
pared to water. This observation indicates that a triple
836
helical arrangement of the single polymer strains as
secondary structure is not present as it is the case for
Scleroglucan and Schizophyllan. The triplex, stabilized
only by hydrogen bridges, of these homoglucans is
destroyed by the addition of DMSO without cleaving
glycosidic linkages; however, shear viscosity is essentially
decreased (Rau 2005).
Branched β-1,3-/β-1,6-glucans are well known as
biological response modifiers (Bohn and BeMiller 1995).
It was shown for extracellular polysaccharides as Sclero-
glucan and Schizophyllan that molar mass, branching and
molecular structure substantially influence the immunoen-
hancing activity (Kulicke et al. 1997; Mueller et al. 2000;
Wang et al. 2008). Studies dealing with the characterization
of anticomplementary activities shown by biopolymers
extracted from fruiting bodies of T. versicolor revealed that
these biopolymers were composed of three molar mass
fractions: 1,200, 150, and 15 kDa (Jeong et al. 2004). After
quantitative isolation of the different components, the
lowest 15 kDa-fraction with also the highest protein content
(18.5% (w/w)) yielded an essentially lower activity com-
pared to the other fractions. The highest activity was
achieved with the 1,200 kDa-fraction which contained just
4.1% protein and only arabinose besides glucose in a molar
ratio of 1:4.30. Therefore, the authors stated that the
anticomplementary activity was mainly attributed to the
carbohydrate moiety. Cui et al. (2007) showed that EPS
produced by T. versicolor Wr-74 and ATCC 20545
stimulated murine splenocytes to increased production of
the cytokines interleukin (IL)-12 and interferon-γ. The
results indicated that lower levels of ≤0.1 μg mL−1 EPS
generally resulted in higher immune responses than did
higher polymer concentrations. Compared to our results,
they detected similar weight average molecular weight
distribution resulting in 2,100–2,300, 24–26, and 2.7–
3.0 kDa. The largest molecular weight fraction was present
at very low concentrations of 2–3.8% (w/w) compared to
the intermediate molecular weight (13–30% (w/w)) and
smallest molecular weight (66–85% (w/w)) fraction. Immu-
nomodulatory activity was also observed by the addition of
EPS derived from T. versicolor LH 1 (Lin et al. 2008).
Murine RAW264.7 macrophages were treated with EPS
which led to an increase of nitric oxide formation as well as
a stimulation of the cytokines IL-1β, IL-6, and tumor
necrosis factor-α. Transferring these results to the investi-
gated EPS, it should also offer an immunoenhancing effect
and, probably, higher activity because the 4,100-kDa
fraction possesses the preponderate mass proportion of
91% (w/w).
T. versicolor ATCC 200801 secretes larger amounts of
exopolysaccharide (4.1 g L−1) than those reported for other
strains of the same species when synthetic minimal medium
and low-shear bioreactor cultivation are used. Glucose is
Appl Microbiol Biotechnol (2009) 81:827–837
the main component of the polymer besides small amounts
of other pentoses and hexoses but it differs in the detailed
composition of the monosaccharides compared to other
extracellular T. versicolor polysaccharides reported recently
(Cui et al. 2007; Lin et al. 2008). Furthermore, the EPS is
different in the nature of the glycosidic linkage, composi-
tion of monosaccharides, protein content, and weight-
average molecular weight compared to the well-known
PSK and PSP. The main or core EPS is composed of β-1,3/
β-1,6-linked D-glucose molecules which is identical with
Schizophyllan. This structure provides the origin of the
immunoenhancing activities of many fungal extracellular
polysaccharides. Thus, the additional detected pentoses and
hexoses beside glucose are probably not essential for the
action as biological response modifier.
Acknowledgments The strain T. versicolor ATCC 200801 was
kindly provided by Prof. Dr. Kolonkaya and Dr. Candan, Hacettepe
University, Ankara, Turkey.
References
Aktas N, Cicek H, Unal AT, Kibarer G, Kolankaya N, Tanyolac A
(2001) Reaction kinetics for laccase-catalyzed polymerization of
1-naphthol. Bioresour Technol 80:29–36
Anumula KR, Taylor PB (1992) A comprehensive procedure for
preparation of partially methylated alditol acetates from glyco-
protein carbohydrates. Anal Biochem 203:101–108
Bartnicki-Garcia S (1999) Glucans, walls, and morphogenesis: on the
contributions of J. G. H. Wessels to the golden decades of fungal
physiology and beyond. Fungal Genet Biol 27:119–127
Bock K, Thøgersen H, Webb GA (1983) Nuclear magnetic resonance
spectroscopy in the study of mono- and oligosaccharides. ANN R
NMR S 13:1–57
Bohn JA, BeMiller JA (1995) 1,3-β-D-glucans as biological response
modifiers: a review of structure-functional activity relationships.
Carbohydr Polym 28:3–14
Chaplin MF (1982) A rapid and sensitive method for the analysis of
carbohydrate components in glycoproteins using gas–liquid-
chromatography. Anal Biochem 123:336–341
Cheng GY, Wu GR, Zhou YZ, Cheng SL (1998) Extraction and
characterization of proteoglucan from the mycelium of Poly-
stictus versicolor (L.) Fr. by submerged culture. Zhiwu Ziyuan
Yu Huanjing 7:19–23
Cui J, Chisti Y (2003) Polysaccharopeptides of Coriolus versicolor:
physiological activity, uses, and production. Biotechnol Adv
21:109–122
Cui J, Goh KKT, Archer R, Singh H (2007) Characterisation and
bioactivity of protein-bound polysaccharides from submerged-
culture fermentation of Coriolus versicolor Wr-74 and ATCC-
20545 strains. J Ind Microbiol Biotechnol 34:393–402
Domon D, Costello CE (1988) A systematic nomenclature for
carbohydrate fragmentations in FAB-MS/MS spectra of glyco-
conjugates. Glycoconj J 5:397–409
Gura E, Rau U (1993) Comparison of agitators for the production of
branched β-1,3-D glucans by Schizophyllum commune. J
Biotechnol 27:193–201
Hotta T, Enomoto A, Yoshikumi C, Ohara M, Ueno S (1981) Protein-
bound polysaccharides. US patent 4271151
Appl Microbiol Biotechnol (2009) 81:827–837
Jeong SC, Yang BK, Ra KS, Wilson MA, Cho Y, Gu YA, Song CH
(2004) Characteristics of anti-complementary biopolymer extracted
from Coriolus versicolor. Carbohydr Polymers 55:255–263
Kawagoe M, Hyakumura K, Suye S-I, Miki K, Naoe K (1997)
Application of bubble column fermentors to submerged culture
of Schizophyllum commune for production of L-malic acid. J
Ferment Bioeng 84:333–336
Kim SW, Hwang HJ, Park JP, Cho YJ, Song CH, Yun JW (2002)
Mycelial growth and exo-biopolymer production by submerged
culture of various edible mushrooms under different media. Lett
Appl Microbiol 34:56–61
Kulicke WM, Lettau AI, Thielking H (1997) Correlation between
immunological activity, molar mass, and molecular structure of
different 1,3-β-D-glucans. Carbohydr Res 297:135–143
Lin FY, Lai YK, Yu HC, Chen NY, Chang CY, Lo HC, Hsu TH
(2008) Effects of Lycium barbarum extract on production and
immunomodulatory activity of the extracellular polysaccharopep-
tides from submerged fermentation culture of Coriolus versicolor.
Food Chem 110:446–453
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein
measurement with the folin phenol reagent. J Biol Chem
193:265–275
Manzoni M, Rollini M (2001) Isolation and characterization of the
exopolysaccharide produced by Daedalea quercina. Biotechnol
Lett 23:1491–1497
Maziero R, Cavazzoni V, Bononi VLR (1999) Screening of
basidiomycetes for the production of exopolysaccharide and
biomass in submerged culture. Rev Microbiol 30:77–84
Mueller A, Raptis J, Rice PJ, Kalbfleisch JH, Stout RD, Ensley HE,
Browder W, Williams DL (2000) The influence of glucan polymer
structure and solution conformation on binding to (1–>3)-β-D-
glucan receptors in a human monocyte-like cell line. Glycobiol
10:339–46
Muenzberg J, Rau U, Wagner F (1995) Investigations to the
regioselective hydrolysis of a branched β-1,3-glucan. Carbohyd
Polym 27:271–276
Ng TB (1998) A review of research on the protein-bound polysac-
charide (polysaccharopeptide, PSP) from the mushroom Cor-
iolus versicolor (basidiomycetes: Polyporaceae). Gen Pharmacol
30:1–4
Nimtz M, Mort A, Domke T, Wray V, Zhang YX, Qiu F, Coplin D,
Geider K (1996) Structure of amylovoran, the capsular exopoly-
saccharide from the fire blight pathogen Erwinia amylovora.
Carbohydr Res 287:59–76
Nimtz M, Wray V, Domke T, Brenneke B, Haussler S, Steinmetz I
(1997) Structure of an acidic exopolysaccharide of Burkholderia
pseudomallei. Eur J Biochem 250:608–616
Rau U (1997) Biosynthese, Produktion und Eigenschaften von
extrazellulären Pilz-Glucanen. Shaker, Aachen
Rau U (1999) Production of Schizophyllan. In: Bucke C (ed) Methods
in biotechnology, vol 10: carbohydrate biotechnology protocols.
Humana, Totowa, pp 43–57
837
Rau U (2005) Schizophyllan. In: Steinbüchel A, Doi Y (eds)
Biotechnology of biopolymers, vol 1: from synthesis to patents.
Wiley, Weinheim, pp 703–735
Rau U, Brandt C (1994) Oxygen controlled batch cultivations of
Schizophyllum commune for enhanced production of branched
β-1,3-glucans. Bioproc Biosys Eng 11:161–165
Rau U, Wagner F (1987) Non-Newtonian flow behaviour of colloid-
dispers glucan solutions. Biotechnol Lett 9:95–100
Sakagami H, Aoki T, Simpson A, Tanuma S (1991) Induction of
immunopotentiation activity by a protein-bound polysaccharide,
PSK (review). Anticancer Res 11:993–999
Schilling BM, Rau U, Maier T, Fankhauser P (1999) Modeling and
scale-up of the unsterile scleroglucan production process with
Sclerotium rolfsii ATCC 15205. Bioproc Biosys Eng 20:195–
201
Schilling BM, Henning A, Rau U (2000) Repression of oxalic acid
biosynthesis in the unsterile scleroglucan production process
with Sclerotium rolfsii ATCC 15205. Bioproc Biosys Eng
22:51–55
Sietsma JH, Sonnenberg AMS, Wessels JGH (1985) Localization by
autoradiography of synthesis of (1–3)-β- and (1–6)-β linkages in
a wall glucan during hyphal growth of Schizophyllum commune.
J Gen Microbiol 131:1331–1337
Stephan D (1986) Bildung von β-1,3-Glucanen aus Mono-, Di- und
Polysacchariden mit Sclerotium rolfsii und deren physikochemi-
sche Charakterisierung. Ph.D. thesis, Technical University
Braunschweig, Braunschweig
Taspinar A, Kolankaya N (1998) Optimization of enzymatic chlorine
removal from kraft pulp. Bull Environ Contam Toxicol 61:15–
21
Tavares A, Agapito M, Coelho M, Lopes da Silva J, Barros-Timmons
A, Coutinho J, Xavier A (2005) Selection and optimization of
culture medium for exopolysaccharide production by Coriolus
(Trametes) versicolor. World J Microbiol Biotechnol 21:1499–
1507
Teng HY, Zhang X, Wang B, Zhou YF (2007) Structural analysis of
polysaccharide isolated from Coriolus unicolor. Chinese Pharm J
42:1059–1062
Wang Z, Guo Y, Yuan J, Zhang B (2008) Effect of dietary β-1,3/1,6-
glucan supplementation on growth performance, immune response
and plasma prostaglandin E2, growth hormone and ghrelin in
weanling piglets. Asian Austral J Anim Sci 21:707–714
Yesilada O, Sik S, Sam M (1998) Biodegradation of olive oil mill
waste water by Coriolus versicolor and Funalia trogii: effects of
agitation, initial COD concentration, inoculum size and immobi-
lization. World J Microbiol Biotechnol 14:37–42
Yu-Wen L, Xiong Y-H, Yao Z, Cai W (2006) Studies on the ultrasonic
extraction of Coriolus versicolor polysaccharides. Jiangsi Science
4:303–309
Zhang ZS, Han WW, Pan YJ (2001) Studies on chemical structure of
polysaccharide from fruit body of Coriolus versicolor. Yaoxue
Xuebao 36:664–667