Research J ournal of Biotechnology Vol.

8 (4) April (2013)

Res. J. Biotech
(11)

S Sc cr re ee en ni in ng g o of f a a h hi ig gh h y yi ie el ld d p po ol ly ys sa ac cc ch ha ar ri id de e s st tr ra ai in n f fr ro om m t te en n
e ed di ib bl le e a an nd d m me ed di ic ci in na al l f fu un ng gi i a an nd d o op pt ti im mi iz za at ti io on n o of f i it ts s
c cu ul lt tu ur re e c co on nd di it ti io on ns s
Duan Yan-Qing
2
, Xing Zhan-Chang
3
and Xu Jun-Wei
1
*
1. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, 650500, CHINA

2. Hongyun Honghe Tobacco (Group) Co. Ltd., Kunming, 650202, CHINA
3. Laboratory for Conservation and Utilization of Bio-Resources & Key Laboratory for Microbial Resources of the Ministry of Education,
Yunnan University, Kunming, 650091, CHINA
*xjuwei@163.com

Abstract
Ten edible and medicinal fungi were studied for their
abilities to produce polysaccharide in submerged
culture. Strain specific variation in intracellular and
extracellular polysaccharide production was
quantified under laboratory conditions. Among the
fungi examined, Agrocybe aegirita Mo-Aa was found
to produce high amount (3.77 g/L) of total
polysaccharide. Furthermore, optimization of
production conditions for A. aegirita Mo-Aa
polysaccharide was conducted in submerged culture.
Production of A. aegirita Mo-Aa polysaccharide was
optimal at pH 5.0, an inoculation size of 480 mg/L,
culture period of 9 days and mushroom fermentation
medium. Under these optimal conditions, the
maximum production of A. aegirita Mo-Aa
polysaccharide reached 5.46 g/L. The results obtained
will be useful for enhanced production of A. aegirita
Mo-Aa polysaccharide on a large scale.

Keywords: Agrocybe aegirita Mo-Aa, medicinal fungi,
polysaccharide, yield, optimization.

Introduction
Bioactive polysaccharides are produced by a variety of
microorganisms including bacteria
1
, yeast
2
and fungi
3
.
They have been extensively studied for their
biotechnological applications in health, food and medicine
sectors. Especially, those extracellular polysaccharide
(EPS) and intracellular polysaccharide (IPS) from edible
and medicinal fungi have attracted a particular attention for
their pharmacological activities in the aspects of
hypoglycemia, antioxidant, immuno-stimulating, anti-
tumor, anti-inflammatory and free radical scavenging
properties
4-8
. However, in spite of the benefits of fungi,
polysaccharide industrial production has been limited due
to low production yield.

To achieve higher yield of fungal polysaccharide, it is pre-
requisite, to select one strain with hyper-production of
polysaccharide. Previous studies have found that various
edible and medicinal fungi produce polysaccharide such as
Paecilomyces sp.
9-11
, Ganoderma lucidum
12-13
, Flammulina
velutipes
14
, Grifola frondosa
15-16
and Agrocybe chaingu
17
.
Although it was reported that polysaccharide production
yields and cell growth may vary with respect to fungal
species and culture conditions
9, 17
, there is little information
available regarding comparison of polysaccharide
production among different fungal species.

Because solid cultivation of fungi is a time-consuming and
labor-intensive process, submerged cultivation is viewed as
a promising alternative for efficient production of
polysaccharide from edible and medicinal fungi
18,19
. The
production of IPS and EPS is strongly influenced by the
submerged culture medium and culture conditions such as
culture period, pH and inoculation density
9-23
. Therefore,
optimization of culture conditions is essential for the
enhancement of polysaccharide production in fungi. In the
present study, we screened the maximum polysaccharide
producing strain from ten edible and medicinal fungi. Then,
the polysaccharide production of the strain was enhanced
by optimization of nutritional and environmental conditions
in shake flask culture. The information obtained is
considered fundamental and useful for the efficient
production of fungal polysaccharide on a bioreactor scale.

Material and Methods
Organisms and culture conditions: Ten edible and
medicinal fungi (Table 1) were obtained from Laboratory
for Conservation and Utilization of Bio-Resources, Yunnan
University. All strains were maintained on potato dextrose
agar slant and the slants were incubated at 28
o
C for 7 days
and then stored at 4
o
C for about 2 weeks. Preculture
medium consisted of the following components (g/liter):
glucose (35), peptone (5), yeast extract (2.5), KH
2
PO
4
.H
2
O
(1), MgSO
4
.7H
2
O (0.5) and vitamin B1 (0.05). For the first-
stage pre-culture, 40-ml medium with an initial pH of 5.5
was prepared in a 250-ml flask and then 10-ml mycelium
suspension from a slant culture was inoculated and
followed by 5-day incubation at 28
o
C on a rotary shaker
(120 rpm). For the second-stage pre-culture, 45-ml medium
was prepared in a 250-ml flask and inoculated with 5-ml
first-stage pre-culture broth (with ca. 100 mg dry cell
weight per liter (DCW/L)), then followed by 3-day
incubation at 28
o
C on a rotary shaker (120 rpm).

For the shake-flask fermentation, a 45-ml mushroom
fermentation medium (MFM) in a 250-mL flask was
inoculated with 5 ml of second-stage pre-culture broth
(with about 300 mg DCW/L). The culture was incubated in
the dark at 28
o
C on a rotary shaker at 120 rpm. MFM
Research J ournal of Biotechnology Vol. 8 (4) April (2013)
Res. J. Biotech
(12)

consisted of the following components (g/liter): glucose
(35), peptone (5), yeast extract (5), KH
2
PO
4
H
2
O (1),
MgSO
4
7H
2
O (0.5) and vitamin B1 (0.05).

Determination of dry cell weight, EPS and IPS:
Mycelia were harvested by centrifuging a sample at 10,
000 X g for 10 min and the precipitated cells were
washed for three times with distilled water and then
dried at 50
o
C for sufficient time to constant weight. The
dry cell weight was measured by the gravimetric
method. EPS and IPS were extracted and measured
according to the method described by Tang and Zhong
12
.
For all the experimental data, data are the averages of
three independent sample measurements. The error bars
indicate the standard deviations from the mean of
triplicates.

Optimization of culture conditions for polysaccharide
production: The effect of culture medium on A. aegirita
Mo-Aa polysaccharide production was studied by using
four different media. Four different culture media; i.e.
mushroom fermentation medium (MFM), mushroom
complete medium (MCM), yeast malt extract medium
(YM) and potato malt peptone medium (PMP), were
employed to select a suitable medium for polysaccharide
production and mycelia growth. MCM medium consisted
of the following components (g/liter): glucose (20), peptone
(2), yeast extract (2), KH
2
PO
4
(0.46), K
2
HPO
4
(1) and
MgSO
4
(0.5). YM medium consisted of the following
components (g/liter): glucose (10), peptone (3), yeast
extract (3) and malt extract (3). PMP medium consisted of
the following components (g/liter): malt extract (10),
peptone (1) and potato dextrose broth.

The effects of culture period on IPS and EPS production
were investigated. For the shake-flask cultivation of A.
aegirita Mo-Aa, a 45-ml culture medium in a 250-ml flask
was inoculated with 5 ml of second-stage pre-culture broth
(with about 300 mg DCW/L). The culture was incubated in
the dark at 28
o
C on a rotary shaker at 120 rpm. Biomass,
IPS production and EPS production were measured after 0,
3, 5, 7, 9, 11, 13 and 15 days of fermentation. The effects
of inoculation density on the cell growth and production of
IPS and EPS by A. aegirita Mo-Aa were studied by
controlling inoculum sizes at 120, 240, 360, 480 and 600
mg DCW/L. After cultivation, samples were taken for
analysis of biomass, IPS production and EPS production.

The effects of initial pH on the cell growth and production
of IPS and EPS by A. aegirita Mo-Aa were investigated by
setting different initial pH values. Before sterilization, the
medium pH was adjusted to 4, 5, 6, 7 and 8 by adding 1 N
HCl or 1 N NaOH and the corresponding culture pH of 4,
5, 6, 7 and 8 was obtained after inoculation with the
second-stage pre-culture broth. Biomass, IPS production
and EPS production were analyzed after cultivation.

Results and Discussion
Screening of a strain with hyper-production of
polysaccharide from ten edible and medical fungi: The
experiment results (Table 1) indicated that all ten edible
and medical fungi produced variable amounts of IPS and
EPS. As shown in table 1, IPS production was a main
contributor to the total polysaccharide level in all tested
fungi. Among the fungi studied, A. aegirita Mo-Aa was
found to produce the highest amount of intracellular and
total polysaccharide, 3.61 g/L and 3.77 g/L. In the earlier
studies reported, G. lucidum was found to produce the
highest amount of IPS, 2.22 g/L, grown in the optimal
culture medium
24
. In submerged cultivation of G. frondosa,
the higher IPS production (0.25 g/L) was found in 4%
glucose media with 0.5% (v/v) plant oil addition on day 3
25
.
In the present investigation IPS (3.61 g/L) produced by A.
aegirita Mo-Aa was 1.6 times higher than results obtained
by G. lucidum and 14 times higher than results obtained by
G. frondosa. Thus, A. aegirita Mo-Aa was identified as a
potential IPS producing strain. The culture conditions of A.
aegirita Mo-Aa were further optimized for enhancement of
polysaccharide production.

Effect of different culture media on production of
polysaccharide by A. aegirita Mo-Aa: Nutritional status
showed great influence on cell growth and polysaccharide
production in submerged cultivation of fungi
9,26
. Four
different media, i.e. MCM, MFM, YM and PDP medium
which have usually been used for the cultivation of higher
fungi
17, 27
, were employed to find a suitable culture medium
for A. aegirita Mo-Aa polysaccharide production. In
comparison of MCM, YM and PDP medium, MFM
medium was favorable for both mycelia growth and
polysaccharide production. As shown in table 2, the
maximum biomass, IPS and EPS production were achieved
in MFM medium at 10 d of fermentation of A. aegirita Mo-
Aa. The total polysaccharide production (3.72 g/L) was
obtained in MFM medium which was 1.3, 4.9 and 1.7 time
higher than those in MCM, YM and PDP medium
respectively. From this result, MFM medium was chosen
for further experiments to screen the culture conditions for
polysaccharide production. Our results also showed that
IPS production of A. aegirita Mo-Aa may be associated
with mycelia growth. Similar results were also reported in
Agaricus blazei AB2003
28
. In Corious versicolor, it was
reported that mycelia growth was closely related to
polysaccharide production
29
.

Effect of cultivation period on production of
polysaccharide by A. aegirita Mo-Aa: Figure 1 shows
dynamics of cell growth, IPS and EPS production in
submerged culture of A. aegirita Mo-Aa. Fungal growth
increased until the 9th day and then remained
approximately constant thereafter. The production of IPS
achieved maximum of 3.75 g/L after 9 days of cultivation
and then decreased slowly. For EPS production, it
increased slightly during the whole cultivation and reached
a maximal value of 0.55 g/L at the end of cultivation. The
Research J ournal of Biotechnology Vol. 8 (4) April (2013)
Res. J. Biotech
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maximal A. aegirita Mo-Aa total polysaccharide
production (4.15 g/L) was achieved at 9th day of
cultivation.

Figure 1: Time profiles of biomass (open circles), IPS
production (open squares) and EPS production (open
triangles) in MFM medium during the submerged
cultivation of Agrocybe aegirita Mo-Aa.

Effect of inoculation density on production of
polysaccharide by A. aegirita Mo-Aa: Inoculation density
is an important factor for the submerged fermentation of
many fungi
23, 30, 31
. To examine the effect of inoculation
density on polysaccharide production, A. aegirita Mo-Aa
was inoculated in MFM medium varying the inoculate size
(120, 240, 360, 480, 600 mg DCW/L). Table 2 clearly
shows control of inoculation density affected IPS and EPS
production in cultivation of A. aegirita Mo-Aa. The
production of IPS at an inoculation size of 120, 240, 360,
480 and 600 mg DCW/L was 3.00, 3.25, 3.76, 4.52 and
4.04 g/L, respectively. The production of EPS at an
inoculation size of 120, 240, 360, 480 and 600 DCW/L was
0.44, 0.45, 0.26, 0.50 and 0.44 g/L respectively. These
results indicated that a high production on IPS and EPS
could be achieved at a relative large inoculum size in the
submerged culture of A. aegirita Mo-Aa. It was also
reported that a higher IPS production was obtained at a
large inoculum size in the G. lucidum and Tuber sinense
fermentation. The maximal G. lucidum IPS production of
1.22 g/L was obtained at a large inoculation density of 670
mg DCW/L as investigated
30
. The T. sinense maximum IPS
production of 1.40 g/L was obtained at an inoculation level
of 487 mg DCW/L
23
. Our results indicated that control of
inoculation density was significant for cell growth and the
production of EPS and IPS and an inoculation density of
480 mg DCW/L were favorable for the submerged
cultivation of A. aegirita Mo-Aa.

Effect of pH on production of polysaccharide by A.
aegirita Mo-Aa: The initial pH of culture medium is an
important factor affecting cell membrane function, the
uptake of various nutrients and product biosynthesis
32
. The
influence of initial pH on production of A. aegirita Mo-Aa
polysaccharide was investigated in the range of pH 4-8. As
shown in table 2, growth of A. aegirita Mo-Aa and
production of polysaccharide was affected by initial pH.
The maximum IPS and EPS production was 4.93 and 0.53
g/L respectively with an initial pH of 5.0. Generously,
many kinds of fungi prefer more acidic conditions optimal
to achieve submerged culture
33-36
. It has also been reported
that the optimal culture pH for biomass and polysaccharide
production by a few fungi occurred with medium or high-
medium culture pHs
37-38
. Our results showed that the
optimal culture initial pH for A. aegirita Mo-Aa was 5.0. A
previous report showed that the optimal initial pH for
production of polysaccharide was pH 6.0 in Agrocybe
cylindracea
39
. These are consistent with the fact that many
fungi have acid pH optimal during submerged cultures.

Conclusion
Species screening results identified A. aegirita Mo-Aa as a
potential polysaccharide producing strain whose production
was higher than results obtained by other tested fungi.
MFM medium, culture period of 9 days, inoculation size of
480 mg DCW/L and pH 5.0 were optimal for
polysaccharide production in A. aegirita Mo-Aa. The
maximum of total polysaccharide production (5.46 g/L)
was attained under the optimal culture conditions. This
work will be useful for enhanced production of A. aegirita
Mo-Aa polysaccharide on a large scale.

Acknowledgement
This work was funded jointly by projects from Department
of Science and Technology of Yunnan Province
(2012BA015), CNTC (110201201009 BR-03), Yunnan of
CNTC (2011CP02), Hongyun Honghe Tobacco (Group)
Co. Ltd. (HYHH2012HX0HYHH2012HX05, 2012JC08)
and a start-up grant from Kunming University of Science
and Technology (KKSY201226107).

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Table 1
Comparison of dry cell weight, IPS and EPS production (on day 9) of different mushrooms in MFM medium
Strain Mean valueSD
Dry cell weight IPS production EPS production
(g/L) (g/L) (g/L)
Paecilomyces prinosa 14.710.09 1.570.04 0.500.01
Paecilomyces militaris 9.130.10 0.610.01 0.110.01
Paecilomyces gunnii 14.350.11 0.990.03 0.130.01
Paecilomyces hepialid 12.240.20 1.160.05 0.580.01
Ganoderma lucidum 20.231.40 2.480.10 0.190.01
Coprinus comatus 18.580.22 1.920.08 0.610.02
Strobilomyces floccopus 17.360.18 1.830.08 0.500.02
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Grifola frondosa 9.910.09 1.190.06 0.530.01
Agrocybe aegirita Mo-Aa 14.110.11 3.610.11 0.160.01
Table 2
Effects of different culture conditions on dry cell weight, IPS production and EPS production in cultivation of
Agrocybe aegirita Mo-Aa
Culture conditions Mean value + SD
Dry cell weight IPS production EPS production
(g/L) (g/L) (g/L)
MFM medium 12.130.43 3.180.12 0.540.02
YM medium 11.661.50 2.680.02 0.160.01
MCM medium 3.800.05 0.610.10 0.150.01
PDP medium 6.180.57 1.880.15 0.410.03
Inoculation density (120 mg DCW/L) 10.250.16 3.000.25 0.440.03
Inoculation density (240 mg DCW/L) 11.361.15 3.250.11 0.450.01
Inoculation density (360 mg DCW/L) 14.411.23 3.760.22 0.260.03
Inoculation density (480 mg DCW/L) 13.441.21 4.520.41 0.500.01
Inoculation density (800 mg DCW/L) 12.121.16 4.040.75 0.440.02
Initial pH 4 13.781.01 4.820.34 0.440.03
Initial pH 5 12.420.37 4.930.50 0.530.02
Initial pH 6 11.860.84 4.700.42 0.400.04
Initial pH 7 11.781.13 4.320.38 0.450.01
Initial pH 8 10.980.96 3.900.41 0.420.01

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(Received 20
th
January 2013, accepted 25
th
March 2013)