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Immunostimulatory and Imunomodulant Activities of Extra
and Intracellular Fungal Polysaccharides
of Ganoderma lucidum
by Submerged and Solid State Cultivation
M. Berovic1, J.Habijanic1, B. Boh2, B. Wraber3
1Faculty of Chemistry and Chem. Engineering 2Faculty of Science and Engineering,
3 Medical Faculty
University of Ljubljana, Slovenia
WOOD-DEGRADING MUSHROOMS
White rot Brown rot
Ganoderma lucidum
Ganoderma lucidum
Antiinflammatory
Antiandrogen (in
prostate cancer and
benign prostatic
hyperplasia)
Antimutagenic and
cell protective
Antiangiogenic
Complement
inhibition
Platelet aggregation
Anti-HIV, antiviral
Hypocholesterolemic
Antihepatotoxic and
hepatoprotective
Antihypertensive
Antihistaminic
Antitumor and
anticancer
Effects of
triterpenoids
Ganoderma
lucidum
pharmacological
effects
Effects of
polysaccharides
Analgesic and
antiarthritic
Antidiabetic
Antioxidant
Cell protection
Other immune
functions
Antitumor effect
through
antiangiogenesis
Antitumor effect
through
immunomodulation
F. Pohleven
Ganoderma lucidum
Regnum: FUNGI
Phyllum: EUMYCOTA
Subphyllum: BASIDIOMYCOTINA
Clasis: HYMENOMYCETES
Ordo: APHYLLOPHORALES
Familia: POLYPORACEAE
Genus: GANODERMA
Species: LUCIDUM
Ganoderma species in Slovenia
G. adspersum (Schultz) Donk
G. carnosum Pat.
G. lipsiense (Batsch) Atk. (= G. applanatum)
G. lucidum (Leyss.:Fr.) Karst
G. pfeifferi Bress
G. resinaceum Boud.
G. valesiacum Boud
BIOREACTOROMICS
Submerged cultivation
Ganoderma lucidum
innoculum used in
submerged cultivation
Submerged cultivation
Batch cultivation of Ganoderma lucidum biomass and polysaccharide production
(T = 30C, N = 300 min-1, Qg = 10 L min-1) Biomass ; Δ Extracellular ; ▲ Intracellular polysaccharides
Ganoderma lucidum submerged cultivation
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0
2
4
6
8
10
12
14
0 25 41 65 90 113 143 168 182 208
Ko
nc
en
trac
ija
po
lis
ah
ari
do
v (
g/L
)
Ko
nc
en
tra
cij
a b
iom
as
e (
g/L
)
Čas kultivacije (h)
Biomasa
Zunajcelični polisaharidi
Micelijski polisaharidi
Po
lysacch
ari
des (g
/l)
Bio
mass (
g/l)
Fermetation time ( h)
Influence of feding on Ganoderma lucidum biomass and polysaccharide production in fed
batch cultivation, (T = 30C, N = 300 min-1, Qg = 10 L min-1) 17 % inoculum
Ganoderma lucidum submerged cultivation
0
2
4
6
8
10
12
14
0 200 400 600 800 1000 1200
čas (h)
ko
nc
en
tra
cij
a b
iom
as
e (
g/l
)
1. dohranitev 2. dohranitev 3. dohranitev 4. dohranitev
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 200 400 600 800 1000 1200
čas (h)
ko
nc
en
tra
cij
a p
oli
sa
ha
rid
ov
(g
/l)
1. dohranitev2. dohranitev 3. dohranitev 4. dohranitev
Bio
mass (
g/l)
1.Feed 2.Feed 2.Feed 1.Feed 3.Feed 4.Feed 4.Feed 3.Feed
Fermetation time ( h) Fermetation time ( h)
Po
lysacch
ari
des (g
/l)
Solid state cultivation
Horizontal Stirred Tank Reactor
BIOREACTORS
Ganoderma lucidum
J. Habijanic & M. Berovic
Mycelial growth after 6 weeks
Ganoderma lucidum
Mycelial growth after 6 weeks
J. Habijanic & M. Berovic
Ganoderma lucidum
Mycelial growth after 6 weeks
GROWTH IN SSF
Mycelial growth on solid substrate after 7 days of
cultivation (350x)
GROWTH IN SSF
Mycelial growth on solid substrate after 12 days of
cultivation (350x)
J. Habijanic
GROWTH IN SSF
Mycelial growth on solid substrate after 14 days of
cultivation (350x)
J. Habijanic
Grifola frondosa
Corn cellulose
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 10 20 30 40 50
time (day)
glu
cosam
ine (
mg/g
)
biomass exp. 1
biomass exp. 2
0
1
2
3
4
5
6
0 10 20 30 40 50time (day)
poly
saccharides (
mg/g
)
polysaccharides exp. 1
polysaccharides exp. 2
Biomass growth during both
experiments
Polysaccharide production during
both experiments
Ganoderma lucidum solid state experiments
Ganoderma lucidum biomass and polysaccharide production
(T = 30C, N = 300 min-1, Qg = 10 L min-1) Biomass ; Δ Extracellular ; ▲ Intracellular polysaccharides
Ganoderma lucidum solid state experiments
0
1
2
3
4
5
6
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 7 14 21 28 35 42 49
Ko
nc
en
trac
ija
po
lis
ah
ari
do
v (
mg
/g)
Ko
nc
en
tra
cij
a g
luk
oza
min
a (
mg
/g)
Čas kultivacije (dnevi)
Biomasa - Poskus 3 (neprestano vlaženje)
Micelijski polisaharidi - Poskus 3 (neprestano vlaženje)
Zunajcelični polisaharidi - Poskus 3 (neprestano vlaženje)
Fermetation time ( h)
Bio
mass –
Glu
co
sam
ine c
on
cen
trati
on
(m
g/g
)
Po
lysacch
ari
des (m
g/g
)
Study course MYCELIUM
Vacuum filtration
PRECIPITATION
HOT
WATER
FILTRATE
CULTIVATION
BROTH
1% NH4
OKSALATE
EXTRACTION
5% NaOH
96%
EtOH
96%
EtOH
96%
EtOH
96%
EtOH
ACETIC
ACID
EXTRACT - POLYSACCHARIDES
A B C D E
ION EXCHANGE CROMATOGRAPHY
GEL FILTRATION
A C D
AFFINITY CROMATOGRAPHY
A- C-
B
B-
INTRA CELLULAR
EXTRACELLULAR
E
INFLAMMATORY AND IMMUNOMODULATORY STUDIES
Tested in immuno-
studies
Ion-exchange chromatography Elution chromatogram of a water soluble
fraction of extracellular polysaccharides
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
10
50
90
130
170
210
250
290
330
380
450
490
540
620
670
720
800
860
920
Volume/mL
Abso
rban
ce
0.1M NaHCO30.5M NaHCO3 0.1M NaOH
0.3M NaHCO3
fraction E1
Abso
rban
ce
0
0.5
1
1.5
2
2.5
3
3.5
10
60
110
160
210
260
310
360
410
460
510
560
610
660
710
760
810
860
910
Volume/mL
0.1M NaHCO3
0.3M NaHCO3
0.5M NaHCO3
0.1M NaOH
fraction I1 fraction I2
Ion-exchange chromatography
Elution chromatogram of a water soluble
fraction of intracellular polysaccharides
fraction I1-2
0
0.5
1
1.5
2
2.5
3
3.5
4
10
20
26
32
38
44
50
56
62
68
74
80
86
Volume/mL
Ab
sorb
an
ce
fraction E1
fraction I1
fraction I2
fraction E1-2
Gel filtration chromatography elution chromatograms of
extracellular polysaccharide
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
0 5 10 15 20 25 30 35 40 45 50
Volume / mL
Ab
so
rb
an
ce
Affinity chromatography
on Concanavalin A - Sepharose 4B:
chromatograms of -polysaccharides
Extraction and fractionation of
polysaccharides
• Cultivation broth vacuum filtered
• Extracellular polysaccharides: liquid medium
concentrated, precipitated with 96 % ethanol, filtered,
washed with acetone, dried - Fraction A.
Extraction and fractionation of
polysaccharides
• Intracellular polysaccharides: mycelium pre-extracted
with 85 % ethanol, extraction with hot water, precipitation
by 96 % ethanol - Fraction B
• Mycelium further extracted with 1% ammonium oxalate
solution - Fraction C
• and with 5 % sodium hydroxide solution; polysaccharides
precipitated by acetic acid - Fraction D
• from the remaining solution precipitated by ethanol-
Fraction E.
Ethanol-precipitated amorphous polysaccharides (37x)
Characteristics and yields of polysaccharide fractions from G. lucidum mycelium
Fraction Properties Mass (mg) Yield (%)
A
extracellular polysaccharides,
water soluble,
precipitated with 96% ethanol
1616
1,37
B
intracellular polysaccharides,
hot water extract,
precipitated with 96% ethanol
2414
2,04
C
intracellular polysaccharides,
1% ammonium oxalate solution
extract,
precipitated with 96% ethanol
1183
1,002
D
intracellular polysaccharides,
5% sodium hydroxide solution extract,
precipitated with acetic acid
2068
1,75
E
intracellular polysaccharides,
5% sodium hydroxide solution extract,
precipitated with 96% ethanol
650
0,55
Inflammatory response on Polysaccharides
vs. Polysaccharide-protein complexes
• The induced production of inflammatory cytokine TNF-α was
evaluated and compared both for polysaccharide-protein
complexes and polysaccharides.
• The comparison of the TNF-α inducing capacity of crude
fractions fractionated and purified by ion-exchange
chromatography and gel-filtration
• During our studies it was concluded that concentration 100 g
ml-1 is the most appropriate to study immune responses.
• Following the stimulation of PBMC with concentration 100 g
ml-1, the supernatants were screened for the content of TNF-α
after a 4-h incubation.
Comparison of TNF-α inducing capacity of 5
polysaccharide and polysaccharide-protein
complex fractions
Polysaccharide
fractions (without
proteins) induce
higher amounts of
TNF-α
(up to 2224 pg ml-1)
than polysaccharide-
protein fractions.
The highest difference
observed at cellular
fractions C and B.
The effect of polysaccharides from G. lucidum
on innate inflammatory response
• Polysaccharides (without proteins) have demonstrated higher TNF- inducing capacity versus polysaccharide-protein complexes (demonstrated in previous study) further studies have been focused on :
– effect of polysaccharides on primary inflammatory immune response including cytokine responses: TNF-, IFN- and IL-12
– compare the inducing capacity between polysaccharides obtained after gel filtration and pure -glucans to study the further effect of purity.
• Extracellular (Fraction A) and two Intracellular polysaccharide fractions (Fractions B and C) with highest TNF- inducing capacity (from previous study) have been studied.
• Polysaccharides have been further separated by affinity chromatography on Concavalin A-Sepharose 4B column to obtain purified polysaccharides of -configuration (-glucans) and -configuration (-glucans). Our study was focused on -glucan fractions.
TNF- response
Intracellular
polysaccharides
(Fraction B and C)
have significantly higher
TNF-α inducing capacity
than extracellular
polysaccharides
(Fraction A).
From all fractions the
highest TNF-α inducing
capacity (2413 pg ml-1) is
observed at Fraction B
-glucans.
IFN- response
All -glucan fractions induce
very low levels of IFN-.
All three fractions obtained
after gel filtration demonstrate
capacity below the detection
limit.
B fraction , Intracellular
polysaccharides
demonstrates the highest
capacity among all fractions.
A fraction , Extracellular
polysaccharides
The lowest capacity was
observed .
IL-12 response
Intracellular polysaccharides
(Fraction B and C)
induce higher amounts of cytokine IL-12
Compared with
Extracellular polysaccharides (Fraction A).
Fraction B
The highest IL-12 inducing capacity
(3664 pg ml-1)
followed by Fraction C obtained after gel filtration, compared to inducing capacity of LPS.
Effect of polysaccharides on
primary inflammatory immune
response - summary
Intracellular polysaccharides (Fractions B , C)
induce higher inflammatory response (at all three
cytokines) than extracellular polysaccharides
(Fraction A). The graphical pattern of response is
similar in all three Figures.
Water soluble -glucans extracted by hot water
(Fraction B) demonstrate highest ability to induce
inflammatory response followed by water soluble
polysaccharides extracted by 1 % ammonia
oxalate solution and purified by ion-exchange
chromatography and gel-filtration (Fraction C).
In general -glucan fractions induce stronger
inflammatory response than fractions obtained
after gel filtration. Exceptions are observed at
fractions C in case of TNF- and IL-12 response.
TNF-
IFN-
IL-12
Further study on fraction C on TNF-
inducing capacity
• Based on previous study fraction C demonstrated higher inflammatory
responses on polysaccharides obtained after gel filtration than pure -glucans.
• The fraction obtained after gel filtration is a complex containing
polysaccharides with -configuration (-glucan) and -configuration (-
glucan). During affinity chromatography step those two are being separated.
• In our study the ability of C fraction -glucan has been tested on TNF-
inducing capacity and therefore potential contribution of -glucan to overall
inducing capacity in the complex.
• Following the stimulation of PBMC with C fraction -glucan in concentration
100 g ml-1, the supernatants were screened for the content of TNF- after a
4-h incubation.
TNF- inducing capacity of Fraction C -glucan
-glucan demonstrates
2.5-higher capacity to induce
TNF- response than -
glucan.
-glucan’s higher capacity
might contribute to TNF-
inducing capacity of the
Fraction C complex obtained
after gel filtration.
Th1 vs. Th2 immuno response
Polysaccharides direct lymphocyte response into Th1
• IL-2 and IFN- positive response and
Polysaccharides do not direct lymphocyte response into Th2
• IL-4 negative to neutral response
IL-2
IFN-
IL-4
+ IMMUNOMODULATION WITH
POLYSACCHARIDES A,B,C
POLYCLONAL ACTIVATION:
PMA – Phorbal miristate acetat
IONO – Ionomycine
Conclusions
Original strains of Ganoderma lucidum ( MZKI G97) was isolated
from Slovenian forests.
Submerged and solid state cultivations were applied.
In 14 days Submerged Cultivation fed batch cultivation extracellular (1,7 gl-1) and intracellular (0.45 gl-1) polysaccharide fractions were isolated, up to 17.0 gl-1dry fungal biomass was
produced.
In in 18 days solid state cultivation extracellular (5.77 mg /g ) of and intracellular ( 1.45 mg /g)
polysaccharide was produced at the end of the cultivation.
Isolation of fungal polysaccharides
Polysaccharides were further separated by ion-exchange, gel and affinity chromatography.
The isolated polysaccharides were mainly -D-glucanes.
Immunostimulatory effects of isolates were tested on induction of cytokine
(TNF- , IFN- and IL12) synthesis
Summary of findings
Inflammatory response:
• Intracellular polysaccharides induce higher inflammatory response than extracellular
polysaccharides. The highest response is observed with hot water extracted -glucans.
• Polysaccharides have higher TNF- inducing capacity than polysaccharide-protein
complexes.
• Pure -glucans induce higher inflammatory response (observed with all cytokines: TNF-
, IFN- and IL-12) then polysaccharides obtained after gel filtration. -glucan could
contribute to response of fraction C, which demonstrates opposite results.
• The graphical pattern of response is similar among all three cytokines, however IFN-
response is very low or below detection limit.
Immunomodulation:
• Extracellular polysaccharides have higher response on lymphocyte immunomodulation
than intracellular polysaccharides. The highest effect is observed with fractions obtained
after gel filtration.
• Polysaccharides direct lymphocyte response into Th1 reponse.
Research Team
Faculty of Chemistry
and Chem.Eng.
Prof.Dr.Marin Berovic
Mirjan Svagelj
Irena Zore
Jozica Habijanic
Jasna Potocar
Tanja Blazic
Masa Kodela
David Voglar
Faculty of Natural
Sciences and Tech.
Prof.Dr.Bojana Boh
Damjan Hodzar
Jure Vizijak
Biotechnical Faculty
Prof.Dr.Franc Pohleven
Prof.Dr.Kristina Sepcic
Andrej Gregori
Faculty of Pharmacy
Prof.Dr.Borut Strukelj
Jure Pohleven
Medicine Faculty
Prof.Dr.Branka Wraber
Saša Simcic
Research Team Prof.Dr.Marin Berovic
China Team
Instit
ute of Edible Fungi
Shanghai
Prof.Dr.Jingsong Zhang
Prof.Dr.Minjie Chen
Yen Yan
Jai Waia
Medical Health
Centre
Bejing
Prof.Dr.Zhi-bin Lin
Death is not a problem, only life it is...
To be in life, it means to look for the problems…..
Zorba The Greek
Summary of findings (SLO)
Vnetje:
• Vnetje mocneje inducirajo polisaharidi celicne stene. Najvisji odziv
opazen pri beta-glukanih ekstrahiranih z vroco vodo.
• Polisaharidi (brez proteinov) mocneje inducirajo sintezo TNF- kot
polisaharid-proteinski kompleksi.
• Cisti polisaharidi (beta-glukani) mocneje inducirajo vnetje kot
polisaharidi po ciscenju z gelsko filtracijo. C frakcija je izjema, kjer je
mozno da alfa del frakcije prispeva k odzivu kompleksa (iz gelse
filtracije).
• Odzivi na TNF- in IL-12 so zelo podobni, na IFN- so odzivi zelo
nizki oziroma pod mejo detekcije.
Imunomodulacija:
• Limfocitne odzive mocneje modulirajo ekstracelularni polisaharidi.
Navisji odziv opazen pri frakcijah ociscenih z gelsko filtracijo.
• Polisaharidi ojacijo Th1 imunski odziv. T celice
