JOURNAL OF MEDICINAL FOOD

J Med Food 16 (11) 2013, 1004–1012

# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2012.0267

Production of Bioactive Compounds

with Antitumor Activity Against Sarcoma 180 by Pleurotus sajor-caju
Ivaneliza Simionato Assis,1 Mariane Bonatti Chaves,2 Marcia Luciane Lange Silveira,2
Regina Maria Miranda Gern,1,2 Elisabeth Wisbeck,2 Agenor Furigo Júnior,3 and Sandra Aparecida Furlan1,2
1
Master’s Program in Health and Environment and 2Department of Chemical Engineering,
University of the Joinville Region (Univille), Joinville, Santa Catarina, Brazil.
3
Department of Chemical Engineering, Federal University of Santa Catarina (UFSC/CTC/EQA),
Florianópolis, Santa Catarina, Brazil.

ABSTRACT This work studied the influence of culture medium composition and pH on exopolysaccharide (EPS) production
by Pleurotus sajor-caju and validates the antitumor activity of the produced EPSs and of the mycelial biomass (intracellular
polysaccharides [IPSs]) against Sarcoma 180 (S180) cells. The effect of the initial concentrations of (NH4)2SO4, yeast extract and
soy peptone on EPS production by P. sajor-caju was studied in shake flasks. A bioreactor was used to evaluate the pH values and
the initial CaCO3 and glucose concentrations. Extracts of EPSs (PE1) and IPSs obtained through two different separation
processes (PM1 and PM2) were tested on mice inoculated with S180 cells. A medium containing 2.5, 1.0, and 1.0 g/L of
(NH4)2SO4, yeast extract and soy peptone, respectively, provided the highest EPS concentration (0.6 g/L). The use of pH 4.0,
1.0 g/L CaCO3 and 20 g/L initial glucose concentration enhanced EPS productivity (3.84 g/L per hour). The PE1 extract
promoted the highest reduction of S180 growth (86%), followed by the PM2 extract (80%); growth reduction was dose-
independent for both substances. This work provides information about culture medium and conditions that enhanced the
production of extracellular polysaccharides by P. sajor-caju. The results can contribute to the search for new bioactive products
bringing novel aspects to the medical and pharmaceutical areas.

KEY WORDS:  basidiomycetes  bioprocess  polysaccharides

INTRODUCTION
F ungi of the Pleurotus genus, also known as ‘‘oyster
mushrooms,’’ have gained prominence due to their nu-
tritional and medicinal values,1,2 particularly their antitumor
activity.2–4 The medicinal properties of these fungi appear to
be linked to polysaccharides, more specifically b-d-glucans,
which are present in the basidioma cell wall, mycelium and
in the culture broth from submerged cultures.5–7 However,
the success of b-glucans and other mushroom carbohydrate
polymers application requires active research in addressing
the structure–activity relationship,8–10 which depends on the
extraction methods.11,12
Research has been conducted to identify the best medium
composition and the best cultivation conditions for
exopolysaccharide (EPS) production by various fungi spe-
cies because these parameters vary depending on the genus
and species, composition of the culture medium, and culture
conditions.13–17
Manuscript received 25 October 2012. Revision accepted 19 August 2013.
Address correspondence to: Regina Maria Miranda Gern, PhD, Masters Program in
Health and Environment, University of the Joinville Region—Univille, P.O. Box 246,
Joinville 89219-710, Brazil, E-mail: rgern@univille.br
First phase of this work was designed to study the influ-
ence of culture medium composition [initial concentrations
of (NH4)2SO4, yeast extract, soy peptone, and glucose, as
well as the presence of calcium ions] and pH on the EPS
production by Pleurotus sajor-caju in batch culture. Second
phase of the work has been designed to validate in vivo
antitumor activity against Sarcoma 180 (S180) cells of:
EPSs; water-soluble polysaccharides isolated by hot water
from mycelium biomass and mycelial biomass free of lipids.
MATERIALS AND METHODS
Microorganism and maintenance
P. sajor-caju CCB 019 was obtained from the Center for
Basidiomycete Cultivation of the University of São Paulo.
The strain was grown in wheat dextrose agar (WDA) me-
dium18 and stored under refrigeration (4C).
Evaluation of the nitrogen source: assays in shake flasks
Assays for defining the initial (NH4)2SO4, yeast extract
and soy peptone concentrations were performed in accor-
dance with a 23 factorial design (Table 1). Experiments were
carried out in 500 mL Erlenmeyer flasks containing 100 mL
of culture medium named POL (of polysaccharides) by

1004
 ANTITUMOR ACTIVITY OF EPS FROM PLEUROTUS SAJOR-CAJU 1005

Table 1. The 23 Factorial Design acetone to sample ratio of 3:1 (v/v).22 After 24 h of refrig-
for Evaluation of the Initial Concentrations eration (4C), the samples were centrifuged at 4500 g for
of Culture Medium Components 5 min, and the precipitate was washed twice with an ace-
Level
tone–ethanol–distilled water solution at a ratio of 3:1:1
(v/v/v).19 The washed precipitate was dissolved in 72%
Variables Maximum Minimum H2SO4, and the total sugar content was determined using the
(NH4)2SO4 (g/L) 5 2.5
phenol sulphuric method.23
Yeast extract (g/L) 2 1
Soy peptone (g/L) 1 0.5 Kinetic parameters calculation
The maximum EPS concentration (DPmax, g/L) was cal-
culated using DPmax = (Emax - E0), where E0 and Emax are the
EPS concentrations at the beginning of cultivation and at the
Cavazzoni and Adami19 due to its suitability for the pro-
final process time (t, in hours) when the EPS concentra-
duction of polysaccharides by fungi. The composition of
tion reaches its maximum value, respectively. The yield
POL medium was: 2.0 g/L of MgSO4$7H2O, 1.0 g/L
factor of glucose for EPS (g/g) was calculated using
of K2HPO4, and 20.0 g/L of glucose, The concentrations of
YP/S = DPmax/(S0 - Smax), where S0 and Smax are the glucose
(NH4)2SO4 (2.5 or 5.0 g/L), yeast extract (purchased from
concentrations at the beginning of cultivation and at the final
Oxoid Microbiology Products) (1.0 or 2.0 g/L), and soy
process time (t), respectively. To calculate the global and
peptone (0.5 or 1.0 g/L) in POL medium were varied in
maximum productivity (mg/L per hour), QP = DPmax.1000/t
each experiment (Table 1). The flasks were sterilized,
and QPmax = DPmE.1000/tmE were used, respectively, where
inoculated with two 8 mm diameter agar discs containing
tmE denotes the maximum EPS productivity time and DPmE
mycelium, and incubated at 30C and agitated at a rate of 120
is the EPS concentration at tmE. The same methodology was
per min for 7 days in a B. Braun Certomat U shaker. All
used for calculating the kinetic parameters related to the
experiments were carried out in triplicate.
biomass (DX, YX/S, QX, and QXmax).
Evaluation of the culture medium pH and initial glucose
Bioactive compounds for in vivo antitumor test
and CaCO3 concentrations: assays in bioreactor
The culture broth and the mycelial biomass were sepa-
The assays for defining the pH (3.0, 4.0, or spontaneous
rated by filtration. EPS were obtained using the method
pH evolution), the presence or absence of CaCO3 (1.0 g/L)
described by Pokhrel and Ohga.24 Ethanol PA was added to
and the initial glucose concentration (20 and 60 g/L) were
the culture broth in a 1:4 ratio of culture broth to ethanol
conducted in batch culture in a B. Braun bioreactor, Biostat
(v/v), and left overnight at 4C. The addition of ethanol
B model containing 4 L of modified POL medium [2.5 g/L of
precipitates the compounds of higher molecular weight, as
(NH4)2SO4; 0.2 g/L of MgSO4$7H2O; 1.0 g/L of K2HPO4;
the polysaccharides with biological activity, and the low
1.0 g/L of yeast extract; 1.0 g/L of soy peptone] defined in
molecular weight compounds are dispersed in the ethanolic
the assays in shake flasks. pH was controlled using solutions
solution. The EPS precipitate was separated by centrifugation,
of NaOH 6 M and H3PO4 6 N.
lyophilized, and stored in a sealed flask at room temperature
The inoculum was prepared in 2 L Duran flasks containing
as the first extraction step of exopolysaccharides (PE1).
POL culture medium with the following composition (in
To obtain the first fraction step of mycelium polysac-
g/L): (NH4)2SO4 5.0; MgSO4$7H2O 0.2; K2HPO4 1.0; yeast
charides (PM1), the mycelial biomass was first lyophilized,
extract 2.0; soy peptone 1.0; glucose 20, at an initial pH of
dissolved in 500 mL of distilled water, and then heated to
6.5–7.0.14 The flasks were sterilized, inoculated with all the
80C for 4 h under shaking. The solution was filtered
mycelium obtained from a Petri dish after 7 days growth, and
through a 0.45-lm membrane, and the filtrate was concen-
incubated at 30C and 120 agitations per min for 6 days.20
trated by evaporation. The purpose of this method is to
The best conditions determined in each assay were used
promote the removal of particles that are soluble in hot
in subsequent experiments, in the following order: pH,
water, as the carbohydrates. Ethanol PA was added in a 1:4
CaCO3 and initial glucose concentration. All cultures were
ratio of filtrate to ethanol (v/v), mixed vigorously, and left
incubated at 30C, 300 agitations per min, and 0.25 L per
overnight at 4C. The solution was centrifuged (4500 g for
min air flow, generating an initial volumetric oxygen
20 min), washed with ethanol PA and centrifuged again
transfer coefficient (KLa) of 15 per hour.20 Experiments
under the same conditions. The precipitated polysaccharides
were carried out in duplicate.
with high molecular weight thus obtained, were lyophilized
and stored in a sealed flask at room temperature.
Analytical methods
The second extraction step of mycelium polysaccharides
The biomass concentration was determined by dry weight (PM2) was obtained according to Jose et al.25 The mycelial
at 60C for 48 h. The glucose concentration in the culture biomass was lyophilised, and the lipids were extracted in
medium was determined by the enzymatic glucose-oxydase- Soxhlet with petroleum ether for 8–10 h. Methanol was
peroxidase method.21 EPS was extracted by treating the added to the lipid-free material, which was lyophilized after
fermented broth samples with acetone cooled to 8C, at an methanol evaporation and stored in a sealed flask at room
1006 ASSIS ET AL.

temperature. In this method, both the high molecular weight Table 2. Initial Concentrations of Culture Medium
carbohydrates as the low molecular weight present in the Components for Each Experiment, and the Respective
mycelial biomass are preserved and only the lipid fraction is Concentration of Exopolysaccharides Obtained
removed. This extraction method allows us to observe if (NH4)2SO4 Yeast extract Soy peptone EPS
there is influence of lipids on antitumor activity. Experiments (g/L) (g/L) (g/L) (g/L)

Maintenance of animals 1a 5 2 1 0.39 – 0.04

2 5 2 0.5 0.32 – 0.00
Male Swiss albino mice (Mus musculus; 30 – 5 g) were 3 5 1 1 0.34 – 0.01
obtained from the Paraná Institute of Technology, and kept 4 5 1 0.5 0.28 – 0.01
in the Chronic Treatment Laboratory at Univille throughout 5 2.5 2 1 0.32 – 0.04
the period of the experiment. The mice were housed at 6 2.5 2 0.5 0.31 – 0.06
7 2.5 1 1 0.60 – 0.05
22C – 1C, fed a diet of water and dry pellets of Nuvilab CR1 8 2.5 1 0.5 0.49 – 0.03
(Sogorb) ad libitum, and exposed to 12 h of light per day.26
Initial concentrations were determined using the 23 factorial design. EPS
Maintenance of tumor cells concentrations are given as mean – standard deviation (n = 2).
a
Initial nitrogen source concentrations in POL culture medium proposed by
The S180 cells were obtained from the University of Itajaı́ Cavazzoni and Adami.19
and were used for serial transplants through weekly intra- EPS, exopolysaccharides.
peritoneal injections of tumor suspension in mice.27

Tumor induction Kinetic curves for the bioreactor experiments were gen-
erated using data from duplicates. Kinetic parameters were
Tumor induction was performed subcutaneously in the obtained from the curves using Origin 8.0 PRO software.
back of each mouse in the test and positive control groups at This software was also used to find significant differences
a concentration of 5 · 106 cells/animal.21 between the kinetic parameters using the Tukey’s test at a
5% significance level.
Experimental design Data for in vivo antitumor activity were first analyzed using
Animals were distributed in four groups (n = 5): (1) test the Dixon ‘‘Q’’ test at a 95% confidence level,33 followed by
group (with tumor induction and treated with PM1, PM2, variance analysis (ANOVA) at a 5% significance level.
and PE1, at four doses); (2) positive control group (with
tumor induction and without treatment); (3) control of the RESULTS
substance group (without tumor induction and treated with Culture medium composition study
PM1, PM2, and PE1, at four doses); (4) negative control
group (without tumor induction and without treatment). The Calculated EPS concentration (g/L) based on the 23 fac-
treatment consisted of administering PE1, PM1, or PM2 to torial design is shown in Table 2. Figure 1 shows the effect
mice intraperitoneally (i.p.) for 10 days, starting 24 h after
tumor induction, in daily doses of 3, 10, 30, and 100 mg/kg
animal mass.6,28,29 The mice from the positive control group
received a daily dose of 10 mg/kg 0.01 M phosphate-buff-
ered saline (PBS solution) for 10 days.
Tumor development was evaluated 21 days after tumor
induction26,30,31 by determining the tumor mass (g)32 and
calculating the inhibition rate.29,31 The inhibition rate was
calculated using the ratio [(1 - T)/C] · 100, where T is the
tumor mass of the test group, and C is the tumor mass of the
positive control group.
The experiments were approved by the Ethics Committee of
the University of the Region of Joinville (protocol no. 002/
2008) and were carried out in accordance with current guide-
lines for the Care and Use of Laboratory Animals—Com-
mission on Life Sciences, National Research Council, 1996.

Statistical analysis
Results obtained in shake flasks were evaluated using the
Dixon ‘‘Q’’ test at a 95% confidence level to reject deviant
values.33 The effect of each variable and its interactions on
the tested factors was evaluated by the Pareto test using FIG. 1. Effect of the concentrations (g/L) of (NH4)2SO4 and yeast
Statistica 7.0 software. extract on the exopolysaccharide (EPS) concentration (g/L).
 ANTITUMOR ACTIVITY OF EPS FROM PLEUROTUS SAJOR-CAJU
Table 3. Calculated Effects on the Exopolysaccharides
Concentration Based on the 23 Factorial Design
at a 95% Confidence Level
Variables Effect – standard error
(1) (NH4)2SO4 (g/L) - 0.097125 – 0.017169a
(2) Yeast extract (g/L) - 0.095375 – 0.017169a
(3) Soy peptone (g/L) 0.064875 – 0.017169a
Interaction between (1) and (2) 0.140125 – 0.017169a
Interaction between (1) and (3) 0.004375 – 0.017169
Interaction between (2) and (3) - 0.020875 – 0.017169
Interaction between (1), (2), and (3) 0.029125 – 0.017169
a
Statistically significant effect.
of the initial concentrations (g/L) of (NH4)2SO4, yeast ex-
tract, and soy peptone on the EPS concentration.
Effects of each tested variable on EPS concentration are
presented in Table 3. Data show that (NH4)2SO4 and yeast
extract had a statistically significant negative effect on EPS
concentration, while soy peptone had a positive effect.
When the concentrations of (NH4)2SO4 and yeast extract are
low, the EPS concentration increases: this increase is more
pronounced when the concentration of soy peptone is in
the highest level. Thus, the highest EPS concentration
(0.60 g/L) in the range of the tested concentrations (Fig. 1)
can be obtained by considering significant interactions, and
using low concentrations of (NH4)2SO4 and yeast extract
(2.5 and 1.0 g/L, respectively) with a high concentration of
soy peptone (1.0 g/L) in the culture medium.
Influence of pH on P. sajor-caju EPS synthesis
When cultivation was carried out at pH 3.0, glucose was
not consumed and EPS was not produced for up to 470 h,
when cultivation was interrupted.
Figures 2 and 3 show the kinetic curves for the P. sajor-
caju submerged culture, using pH 4.0 and spontaneous pH,
respectively.
The kinetic profiles in Figure 2 show that total glucose
was consumed in 328 h of cultivation. At 293.7 h, the EPS
1007
concentration stabilized at a maximum value at 0.53 g/L,
corresponding to a biomass concentration of 5.52 g/L.
Kinetic profiles in Figure 3 show that the residual glucose
concentration was *1.6 g/L at 340 h, at which time culti-
vation was interrupted. During cultivation, the pH decreased
from an initial value of 7.0 to a stable value of *3.5
in 230 h. At 264.4 h of cultivation, the maximum EPS
concentration and biomass concentration were 0.55 and
3.52 g/L, respectively. However, the biomass production
peaked at 230 h (4.62 g/L), followed by a drop in production
soon after pH stabilization at 3.5. Similar behavior was
observed for EPS production, with a peak in EPS production
in 264.8 h, followed by decreased production.
Table 4 summarizes kinetic parameters obtained for
P. sajor-caju cultivation at pH 4.0 and spontaneous pH.
Much similarity is observed between the EPS concentration
values (DPmax), the yield factor for glucose for EPS (YP/S)
and the global (QP) and maximum productivity for EPS
(QPmax) for both cultures (pH 4.0 and spontaneous pH). The
Tukey’s test produced no statistically significant differences
in kinetic parameters described above.
Influence of CaCO3 on P. sajor-caju EPS synthesis
Figure 4 shows kinetic curves for P. sajor-caju cultivation
at pH 4.0 with addition of CaCO3 (1.0 g/L).
Figure 4 shows that 210 h were required for glucose
consumption when CaCO3 (1.0 g/L) is utilized, compared to
328 h when CaCO3 was not used in the culture (Fig. 2). The
EPS concentration (0.72 g/L) stabilized after 188.4 h, cor-
responding to a biomass concentration of 5.73 g/L (Fig. 4).
Table 3 gives the kinetic parameters for P. sajor-caju cul-
tivation with and without CaCO3 (1.0 g/L). The maximum
EPS concentration was 35.8% higher when calcium car-
bonate was used in the culture medium. The global and
maximum productivity for EPS were also 114.5% and
125.7% higher, respectively, in cultures with added CaCO3
(1.0 g/L). The maximum biomass productivity (QXmax) was
113.3% higher for cultivation without calcium carbonate.
The Tukey’s test does not show any statistically significant
FIG. 2. Profiles of biomass growth: X
(dashed line, -, ,), glucose con-
sumption: S (continuous line, C, B),
and exopolysaccharide concentration:
EPS (dotted line, :, 6) with time (h)
at pH 4.0. The open and closed symbols
represent data from duplicate tests.
1008 ASSIS ET AL.

FIG. 3. Profiles of biomass growth:

X (dashed line, -, ,), glucose con-
sumption: S (continuous line, C, B),
and exopolysaccharide concentration:
EPS (dotted line, :, 6) with time (h)
at spontaneous pH. The open and
closed symbols represent data from
duplicate tests.

differences between cultivations with and without CaCO3 cultivation with 20 g/L initial glucose. No statistically sig-
for other parameters related to the biomass or for the yield nificant differences were found for other kinetic parameters
factor of glucose in EPS and in biomass. related to EPS and the biomass (Table 4).

Influence of the initial glucose concentration In vivo antitumor test

on P. sajor-caju EPS synthesis
Figure 5 shows the kinetic curves for P. sajor-caju cul-
tivation at pH 4.0, with added CaCO3 and an initial glucose
concentration of 60 g/L.
The kinetic profiles in Figure 5 show that the residual
glucose concentration was still *11.4 g/L at 636 h of cul-
tivation, at which time, cultivation was interrupted. How-
ever, for the culture at an initial glucose concentration of
20 g/L (Fig. 4), all the glucose was consumed within a 210-h
period. The EPS concentration (1.45 g/L) stabilized after
535.6 h of cultivation, corresponding to a biomass concen-
tration of 11.43 g/L.
Comparison of the kinetic data for the experiment using
60 and 20 g/L initial glucose has been mentioned in Table 4.
The maximum EPS concentration in cultures with 60 g/L
initial glucose was 101.4% higher than in the culture with
20 g/L initial glucose. However, the global EPS productivity
with 60 g/L initial glucose was 42.2% lower than that for
Figure 6 shows the tumor inhibition rates obtained using
PE1, PM1, and PM2. All tested doses for PE1 and for PM2
showed inhibition rates against S180 cells in relation to the
positive control. A dose of 100 mg/kg of PM1 achieved 80%
tumor reduction, whereas there was no statistically signifi-
cant difference in tumor weight at other doses relative to the
control. The PE1 precipitate achieved the highest reduction
of S180 growth (86%), followed by PM2 extract (82%); the
reduction for both extracts was independent of the dose. The
PM1 extract caused growth reduction (80%) only at a dose
of 100 mg/kg.
DISCUSSION
A culture medium containing 2.5, 1.0, and 1.0 g/L of
(NH4)2SO4, yeast extract and soy peptone, respectively,
increased the EPS concentration, using an initial glucose
concentration of 20 g/L. These results are in agreement with

Table 4. Kinetic Parameters for Pleurotus sajor-caju Cultivation Under Various Experimental Conditions

pH: Spontaneous pH: 4.0 pH: 4.0 pH: 4.0

GC0: 20 g/L GC0: 20 g/L GC0: 20 g/L GC0: 60 g/L
Kinetic data CaCO3: 0 g/L CaCO3: 0 g/L CaCO3: 1 g/L CaCO3: 1 g/L
DPmax (g/L) 0.55 – 0.03 0.53 – 0.01 0.72 – 0.02 1.45 – 0.04
DX (g/L) 3.52 – 0.01 5.52 – 1.17 5.73 – 0.24 11.43 – 4.09
YP/S (g/g) 0.03 – 0.00 0.03 – 0.00 0.04 – 0.01 0.03 – 0.00
YX/S (g/g) 0.21 – 0.01 0.34 – 0.10 0.32 – 0.07 0.25 – 0.06
QP (mg/L per hour) 2.06 – 0.21 1.79 – 0.07 3.84 – 0.30 2.70 – 0.08
QX (mg/L per hour) 13.31 – 0.84 18.78 – 4.26 30.39 – 2.89 21.33 – 7.76
QPmax (mg/L per hour) 2.07 – 0.32 1.87 – 0.10 4.22 – 0.40 3.30 – 0.43
QXmax (mg/L per hour) 18.72 – 2.91 19.57 – 4.20 41.74 – 0.15 28.31 – 7.25

Values are given as means – SD (n = 2).

GC, initial glucose concentration; DPmax, maximum EPS concentration; DX, biomass concentration; YP/S, yield factor of glucose in EPS; YX/S, yield factor of
glucose in biomass; QP, global productivity of EPS; QX, global productivity of biomass; QPmax, maximum productivity of EPS; QXmax, maximum productivity of
biomass.
 ANTITUMOR ACTIVITY OF EPS FROM PLEUROTUS SAJOR-CAJU
results from Confortin et al.,14 who investigated the relative
(NH4)2SO4, yeast extract and soy peptone concentrations in
a culture medium for producing biomass and EPS by
P. sajor-caju. Confortin et al.14 used a factorial design to
show that EPS production was increased by using low
concentrations of (NH4)2SO4 and yeast extract together with
high concentrations of soy peptone in the culture medium.
Organic nitrogen sources are generally better than inorganic
sources for extracellular polysaccharide production in sub-
merged culture of mushrooms. He et al.34 obtained rela-
tively lower EPS production by Lentinus edodes using
inorganic nitrogen sources (0.6–0.8 g/L) than organic ni-
trogen sources (1.0–1.4 g/L). Inorganic nitrogen sources
were not effective for mycelial growth of L. edodes35
and Pleurotus ostreatus.15 Studies on EPS production
by Pleurotus ostreatoroseus and P. ostreatus ‘‘florida’’
by Rosado et al.,5 showed that low concentrations of
(NH4)2SO4 in the culture medium increased EPS production
(5.8 g/L) by the P. ostreatoroseus species. Yeast extract has
often been used to provide necessary growth factors. How-
ever, too high a concentration of yeast extract would lower
the use of other carbon sources and the accumulation of
metabolites.35 Feng et al.35 observed that the biomass and
1009
FIG. 4. Profiles of biomass growth: X
(dashed line, -, ,), glucose consumption: S
(continuous line, C, B), and exopolysac-
charide concentration: EPS (dotted line, :,
6) with time (h) at pH 4.0 with added CaCO3
(1.0 g/L). The open and closed symbols rep-
resent data from duplicate tests.
EPS production by L. edodes significantly increased upon
increasing yeast extract concentration up to about 6.0 g/L,
but decreased sharply beyond this. Comparing experiment 1
(where the medium used was proposed by Cavazzoni and
Adami19) and experiment 7 (Table 1) shows that medium
modifications increased EPS production by P. sajor-caju by
54%. Medium 7 also achieved better homogenization due to
the use of smaller mycelium pellets. Medium 7 was there-
fore, the best of the tested compositions for producing EPS
in the bioreactor.
The use of pH 4.0, 1.0 g/L CaCO3 and 20 g/L initial
glucose concentration increased EPS productivity (3.84 g/L
per hour). The results suggest that the lowest pH of 3.5 may
have a negative influence on biomass and EPS production.
This hypothesis is supported by the results for cultivation at
pH 3.0. Xiao et al.36 have shown that pH affects morphology
and cell structure, as well as altering the function of the
fungal cell membrane. He et al.34 investigated the effect of
initial pH on EPS production by Funalia trogii also called
Trametes trogii. The optimal pH for mycelial growth
(3.5 g/L) was 5.0, whereas the maximum EPS production
(4.0 g/L) was achieved at pH 6.0. Huang and Liu37 opti-
mized submerged cultures by Grifola umbellate. They
FIG. 5. Profiles of biomass growth: X
(dashed line, -, ,), glucose consumption: S
(continuous line, C, B), and exopolysac-
charide concentration: EPS (dotted line, :,
6) with time (h) at pH 4.0 with added CaCO3
(1.0 g/L) and 60 g/L initial glucose. The open
and closed symbols represent data from du-
plicate tests.
1010 ASSIS ET AL.
FIG. 6. Tumor mass (g) and tumor inhibition rate (%) for animals
inoculated with Sarcoma 180 and treated with the following sub-
stances: (A) exopolysaccharide precipitate (PE1); (B) intracellular
polysaccharide (IPS) precipitate (PM1); (C) IPS extract (lipid free)
(PM2). The columns indicate the mean – standard error.
Significant differences are indicated in comparison to *the positive
control group (CP) and **the other groups, using Tukey’s test at a
significance level of 5%.
reported that pH 5.0 yielded the highest EPS concentration
(0.571 g/L), while pH 6.0 yielded the highest biomass
concentration (6.23 g/L). Hwang et al.38 observed that for
Phellinus linteus pH 4.0 was ideal for EPS production
and pH 5.0 was ideal for biomass production: the highest
EPS and biomass concentrations obtained were 3.3 and
*2.7 g/L, respectively. Wisbeck et al.13 studied the influ-
ence of the initial pH of the cultivation medium on EPS and
biomass production by P. ostreatus. They obtained the
highest concentrations of EPS (1.33 g/L) and biomass
(12.86 g/L) with pH 4.0, using the POL medium and 40 g/L
glucose. In contrast, Papaspyridi et al.39 obtained better
results using pH 6.0 in their culture medium optimization for
EPS and biomass production by P. ostreatus. Comparison of
the values obtained in this study with those found in the
literature shows that different cultivation conditions (spe-
cific to each fungal species) are required within the same
genus Pleurotus. The controlled pH (4.0) culture was chosen
in this study considering future cultivation on an industrial
scale because it is known that cultivation under low pH is
less susceptible to contamination by other microorganisms.
In addition, the next stage of this work involves the vali-
dation of CaCO3 use in the medium, and CaCO3 is known to
dissolve more easily at low pH, thereby enabling observa-
tion of culture contamination.
The presence of CaCO3 in the culture medium enhances
the EPS concentration and also the global and maximum
productivity for EPS. However, the maximum EPS con-
centration decreased after 188.2 h. The decline in EPS
concentration was most likely due to the stabilization of the
biomass concentration, as polysaccharides may adhere to
the biomass, making the transport of glucose to the inner cell
difficult, which could stimulate the fungus to excrete b-
glucanase to degrade the polysaccharides and obtain glucose
as proposed by Papagianni.40 However, it was also observed
that the amount of available glucose was very low at this
time, suggesting that the fungus may be degrading EPS to
obtain glucose. Xiao et al.36 attribute the significance of
adding a calcium source to the culture medium to the action
of calcium ions on fungal growth, that is, cell membrane
permeability can be changed via an internal Ca2 + gradient
and by effecting the activity of some fungal enzymes in-
volved in cell wall expansion. In addition, the internal Ca2 +
gradient may inhibit biopolymer synthesis, affecting sugar
and protein contents, resulting in an increase in EPS pro-
duction and inhibiting the accumulation of intracellular
polysaccharides (IPS), that is, stimulating the excretion of
polysaccharides to the outside of the cell.
Usually, EPS concentrations can be increased by using
higher carbon source concentrations in the culture medium.
Huang and Liu37 and Shih et al.16 used higher glucose
concentrations to increase EPS production by Grifola um-
bellata and Grifola frondosa. Increasing the glucose con-
centration from 2% to 4% for G. frondosa, double the EPS
concentration from 0.76 to 1.49 g/L. Wisbeck et al.13 also
obtained a higher EPS concentration and global EPS pro-
ductivity (1.33 g/L and 7.5 mg/L per hour, respectively) with
P. ostreatus by using 40 g/L initial glucose instead of
20 g/L initial glucose. Feng et al.35 obtained a higher EPS
concentration (0.25 g/L) by L. edodes varying glucose
concentration from 0.5% to 3.0%. However, higher glucose
concentrations decreased the EPS concentration. In this
study, 20 g/L (instead of 60 g/L) initial glucose was used
in the culture medium to produce EPS by P. sajor-caju, in
 ANTITUMOR ACTIVITY OF EPS FROM PLEUROTUS SAJOR-CAJU
consideration of productivity and contamination reduction
over long cultivation periods in a large scale production.
Comparing our results for P. sajor-caju with those ob-
tained by Wisbeck et al.13 for P. ostreatus, lower EPS
concentration and productivity values are observed for
P. sajor-caju. Indeed, polysaccharides obtained by the
P. sajor-caju species appear to show higher antitumor
activity. Experiments performed by Dalonso et al.2 found
that polysaccharides extracted from P. sajor-caju fruiting
bodies had a higher reduction rate (85%) in the number of
neoplasic cells (Ehrlich ascites tumor) inoculated in mice
than polysaccharides extracted from P. ostreatus (72%)
fruiting bodies in studies by Wolff et al.4 These studies
motivated this work on maximizing EPS production by
P. sajor-caju and the in vivo validation of P. sajor-caju
activity.
This study shows that the PE1 precipitate promoted the
highest growth reduction in S180, followed by the PM2
extract: the reduction achieved by both these extracts
was dose-independent. The PM1 extract, at a dose of
100 mg/kg, achieved the least growth reduction. Moradali
et al.41 studied the growth inhibiting effect of tumors by
aqueous extracts from various macrofungal species and
observed reductions in tumor growth in S180 varying from
42% to 100%. Fortes et al.42 have suggested that sub-
stances extracted from fungi can modulate carcinogenesis
mainly by stimulating the immunologic system. The results
obtained in this study are in accordance with those found in
literature, showing higher than 79% reduction in tumor
growth. Fan et al.43 obtained a 72.2% reduction in tumor
growth in S180 by treatment with Agaricus brasiliensis
culture broth extracts, which is lower than the reduction rate
obtained in this study. Sarangi et al.44 reported that three
proteoglycan fractions extracted from the P. ostreatus my-
celium promoted an S180 growth reduction of 48%, 76.94%,
and 63.7% (Fractions I, II, and III, respectively) when using
5 mg/kg dose of extracts. Comparing the results of Sarangi
et al.44 with the results of this study, PM2 promoted a higher
growth reduction than PE1 in S180, irrespective of the dose.
Dalonso et al.2 used fractions extracted from the fruiting
body of the same fungus species used in this study
(P. sajor-caju CCB 019) to obtain a 85% reduction in the
number of Ehrlich ascites tumor cells. The extracts were
administered at 10 mg/kg i.p. for six consecutive days. The
chemical characterization of the bioactive compounds
produced was not an objective of this study. However,
there are suggestions in the literature that the composition
of these bioactive compounds includes polysaccharides
and proteins.4,24
ACKNOWLEDGMENTS
The authors thank Dr. A.A. Steil and Dr. D. Sato from
UNIVALI for supplying the tumor strain. We also thank
the Research Support Fund from Univille and the Co-
ordination for the Improvement of Higher Level Educa-
tion Personnel—CAPES/Brazil for the financial support.
1011
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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