Bioresource Technology 96 (2005) 1175–1182

Optimization of submerged culture condition for the production ofmycelial biomass and exopolysaccharides by Agrocybe cylindracea

H.O. Kim a, J.M. Lim a, J.H. Joo a, S.W. Kim a, H.J. Hwang a, J.W. Choi b, J.W. Yun a,*

a Department of Biotechnology , Daegu University, Kyungsan, Kyungbuk 712-714, Koreab Department of Life Resources, Daegu University, Kyungsan, Kyungbuk 712-714, Korea

Received 26 May 2004; received in revised form 20 September 2004; accepted 23 September 2004

Available online 21 November 2004

Abstract

The optimization of submerged culture conditions and nutritional requirements was studied for the production of exopolysac-

charide (EPS) from Agrocybe cylindracea ASI-9002 using the statistically based experimental design in a shake flask culture. Both

maximum mycelial biomass and EPS were observed at 25 �C. The optimal initial pH for the production of mycelial biomass and EPSwere found to be pH4.0 and pH6.0, respectively. Subsequently, optimum concentration of each medium component was determined

using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth was as follows: mal-

tose 80g/l, Martone A-1 6g/l, MgSO4 Æ7H2O 1.4g/l, and CaCl2 1.1g/l; for EPS production: maltose 60g/l, Martone A-1 6g/l,

MgSO4 Æ7H2O 0.9g/l, and CaCl2 1.1g/l. Under the optimal culture condition, the maximum EPS concentration achieved in a 5-l

stirred-tank bioreactor indicated 3.0g/l, which is about three times higher than that at the basal medium.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Agrocybe cylindracea; Exopolysaccharide; Fermentation; Optimization; Orthogonal matrix method; Submerged culture

1. Introduction

During the past decades there has been an increasing

interest in the production of exopolysaccharides (EPS)

from mushrooms due to their various physiological

activities (Chihara et al., 1970; Lee et al., 1996; Maziero

et al., 1999; Rosado et al., 2003; Song et al., 1998). Agro-

cybe cylindracea, which belongs to the class basidiomyce-

tes, has been reported to have beneficial physiological

activities such as anti-tumor (Kiho et al., 1989), hypogly-cemic (Kiho et al., 1994), lipid peroxidation inhibitory

(Lee et al., 1998), and immuno-stimulating activity (Yos-

hida et al., 1996). As a result of its perceived health bene-

fits, A. cylindracea has gained wide popularity as an

effective health food and has become one of the valuable

0960-8524/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2004.09.021

* Corresponding author. Tel.: +82 53 850 6556; fax: +82 53 850

6559.

E-mail address: jwyun@daegu.ac.kr (J.W. Yun).

mushrooms. To date, there are many reports regarding

the culture condition of A. cylindracea, characterizationof polysaccharides and their biological activities pro-

duced from the extract of this mushroom (Lee et al.,

1989; Koshino et al., 1996; Kim et al., 1997, 1999; Ha

et al., 1995; Ngai et al., 2003; Wang et al., 2002).

However, most investigators have made efforts to cul-

tivate this mushroom on solid artificial media rather

than submerged culture. Due to the fact that fruiting

body formation of A. cylindracea has not been devel-oped successfully in solid-state fermentation, the pro-

duction of mycelial biomass by submerged culture

deserves investigating to obtain bioactive materials.

There are large numbers of reports on optimization

of culture media for microbial metabolites by statistical

optimization techniques (Coello et al., 2000; Ergun and

Mutlu, 2000). Orthogonal design is one of the important

statistical methods that use the Taguchi parameterdesign methodology (Montgomery, 1999). It is feasible

1176 H.O. Kim et al. / Bioresource Technology 96 (2005) 1175–1182

to investigate the influence of controlled factors in a

multivariable system using this method. It also can give

effective responses in the course of system optimization.

They have been successfully applied to the improvement

of culture media or the production of primary and sec-

ondary metabolites in the fermentation process (Escam-illa et al., 2000; Lee et al., 1997; Li et al., 2001; Xu et al.,

2003).

This report is an attempt to formulate suitable pro-

duction media for the production of mycelial biomass

and EPS in A. cylindracea using the orthogonal matrix

method. To the best of knowledge, this is the first report

concerning the submerged culture of A. cylindracea.

2. Methods

2.1. Microorganism and media

A. cylindracea ASI-9002 was a culture collection of

our laboratory which had been originally isolated from

a mountainous district in Korea. The stock culturewas maintained on potato dextrose agar (PDA) slants.

The slants were incubated at 25 �C for 7days and then

stored at 4 �C. MCM (Mushroom Complete Medium)

were used as the basal medium. Seed culture was grown

in 250ml flasks containing 50ml of MCM (20g/l glu-

cose, 2g/l meat peptone, 2g/l yeast extract, 0.46g/l

KH2PO4, 1g/l K2HPO4, 0.5g/l MgSO4 Æ7H2O) at 25 �Con a rotary shaker incubator at 150rpm for 8days.

2.2. Inoculum preparation and flask culture

A. cylindracea was initially grown on PDA medium in

a petridish, and then transferred to the seed culture med-

ium by punching out 5mm of the agar plate culture with

a sterilized self-designed cutter. The seed culture was

grown in 250ml flasks containing 50ml of basal mediumat 25 �C on a rotary shaker incubator at 150rpm for

4days. The second flask culture experiments were per-

formed in 250ml flasks containing 50ml of the media

after incubating with 10% (v/v) of the seed culture under

the conditions described above. All experiments were

performed at least in duplicate.

Table 1

Experimental factors and their levels for orthogonal projects

Level A (%) B (%) C (%) D (%)

1 4 0.20 0.04 0.01

2 6 0.40 0.09 0.06

3 8 0.60 0.14 0.11

Symbols A, B, C, and D represent factors of maltose, Martone A-1,

MgSO4 Æ7H2O, and CaCl2. Symbols 1, 2, and 3 represent concentrationlevels of each factor.

2.3. Bioreactor fermentation

Fermentation medium was inoculated with 10% (v/v)

of the seed culture and then cultivated at 25 �C in a 5-lstirred-tank bioreactor (KF-250, KoBioTech, Seoul,

Korea). Fermentations were conducted under the fol-

lowing conditions: temperature 25 �C, aeration rate

2vvm, agitation speed 150rpm, initial pH6.0, and work-

ing volume 3-l. The seed culture was transferred to thefermentation medium and was cultivated for 11days.

2.4. Analytical methods

Samples collected at various intervals from a shake

flask were centrifuged at 9000g for 15min. The resulting

supernatant was filtered through a Whatman filter paper

No. 2 (Whatman International Ltd., Maidstone, UK).The resulting culture filtrate was mixed with four vol-

umes of absolute ethanol, stirred vigorously, and then

left overnight at 4 �C. The precipitated EPS was centri-fuged at 9000g for 15min, and the supernatant was dis-

carded. The precipitate of pure EPS was dried and the

EPS weight was estimated. The mycelial dry weight

was measured after repeated washing of the mycelial

pellets with distilled water and left drying at 70 �C over-night at a constant weight.

2.5. Orthogonal matrix method

The 3k factorial design, a factorial arrangement with

k factors each at three levels, were employed. Factors

and interactions were denoted by capital letters. The

three levels of the factors were set as low, intermediate,and high. Each treatment combination in the 3k designs

was denoted by k digits, where the first digit indicates

the level of the factor A, the second digit indicates the

level of the factor B, and the kth digit indicates the level

of the factor k. For a problem with four design variables

and three levels, the minimum orthogonal matrix

method was selected as L9 (34) as noted in Table 1. Con-

sequently, this enables us to determine which processvariables affect the response. A logical next step is to

determine the point in the important factors that leads

to the best possible response (Montgomery, 1999; Tarng

et al., 2002; Di et al., 2003). The detailed experimental

conditions for each project are listed in Table 2.

3. Results and discussion

3.1. Effect of initial pH and temperature

To find out the optimal temperature for mycelial

growth and EPS production, A. cylindracea was culti-

vated at various temperatures ranging from 15 to

30 �C. Both maximum cell growth and EPS were ob-

served at 25 �C (Fig. 1B), which is comparable to many

Table 2

Application of L9 (34) orthogonal projects to mycelial growth and

exopolysaccharide production by Agrocybe cylindracea ASI-9002

Run A B C D Mycelial

biomass (g/l)

Exopolysaccharides

(g/l)

1 1 1 1 1 3.57 ± 0.48 0.79 ± 0.07

2 1 2 2 2 4.68 ± 0.19 1.42 ± 0.01

3 1 3 3 3 6.54 ± 0.28 2.05 ± 0.27

4 2 1 2 3 7.49 ± 0.73 1.84 ± 0.30

5 2 2 3 2 6.89 ± 0.35 1.58 ± 0.21

6 2 3 1 1 7.67 ± 0.31 1.56 ± 0.08

7 3 1 3 2 8.73 ± 0.25 1.02 ± 0.06

8 3 2 1 3 8.22 ± 0.98 2.08 ± 0.01

9 3 3 2 1 7.72 ± 0.59 1.53 ± 0.07

The arrangements of column A, B, C, and D were decided by

orthogonal design for 4 (factor) · 9 (run number); every row of run

number represents one experimental replicate; every run was carried

out twice. Values are mean ± SD of double determinations.

H.O. Kim et al. / Bioresource Technology 96 (2005) 1175–1182 1177

kinds of mushrooms that have relatively low tempera-

ture optima (e.g. 20–25 �C) in their submerged cultures(Bae et al., 2000; Kim et al., 2003). The pH of medium

is a very important but is often a neglected environmen-

tal factor. Many investigators claimed that the different

morphology of fungi mycelia under a different initial pH

value was the critical factor in biomass accumulation

and metabolite formation (Shu and Lung, 2004; Wangand McNeil, 1995). The medium pH may affect cell

membrane function, cell morphology and structure,

the uptake of various nutrients, and product biosynthe-

sis (Gerlach et al., 1998; Shu and Lung, 2004). In the

present study, maximum EPS concentration (0.72g/l)

was obtained in cultures grown at an initial pH6.0

(Fig. 1A), whereas maximum biomass concentration

(9.76g/l) was obtained at an initial pH of 4.0. However,the difference in EPS production according to the initial

pH was not so significant. It has also been reported that

Initial pH

4 5 6 7 8

yM

ssam

oib l ailec

l/g(

)

0

2

4

6

8

10

12(A)

Fig. 1. Effects of initial pH (A) and temperature (B) on mycelial growth and

ASI-9002: All experimental data were mean ± SD of double determinations.

many kinds of mushroom have more acidic pH optima

for mycelial growth and EPS accumulation during their

submerged cultures. It has been reported that several

kinds of mushroom have more acidic pH optima for

mycelial biomass and EPS accumulation during their

submerged cultures (Kim et al., 2003; Lee et al., 1989;Shu and Lung, 2004).

3.2. Effect of carbon and nitrogen sources

It is generally understood that mycelia of many

mushrooms grow to some extent over a wide range of

carbon source (Yang et al., 2003). To find out the suit-

able carbon source for the EPS production and mycelialgrowth in A. cylindracea, seven carbon sources were

separately provided at 20g/l instead of glucose employed

in the basal medium. Among the carbon sources tested,

glucose yielded the highest mycelial growth (Fig. 2),

whereas maximum EPS production was achieved in

the maltose medium. It is possible that different carbon

sources might have different effects of catabolic repres-

sion on the cellular secondary metabolism. Such a phe-nomenon was also claimed in submerged cultivation of

many kinds of mushrooms (Hwang et al., 2003; Kim

et al., 2003). Nitrogen may be supplied as ammonia, ni-

trate or in organic compounds, such as amino acids and

proteins. Therefore, the omission of nitrogen in the med-

ium greatly affects fungal growth and metabolite pro-

duction. In the course of optimization for nitrogen

sources, individual nitrogen sources were supplementedinto the medium by removing Martone A-1 used in

the basal medium. Fig. 3 shows the effect of nitrogen

sources on mycelial growth and EPS production in a

shake flask culture of A. cylindracea by varying the

nitrogen sources at a concentration level of 4g/l. The

highest mycelial biomass and EPS production were

Temperature (oC)

xE

op

oly

sca

hcirad

es( g

l/)

0

1

2(B)

15 20 25 30

exopolysaccharide production in shake flask cultures of A. cylindracea

yM

ecail

b li

samo

s)l/g(

0

1

2

3

4

5

6

7

poxEylos

hcca( sedirag

)l/

0

1

2

3

4

F urtc o

es

Goculse

caLt so

eaM

tl osenaMn ti

louS

orces

yXol

es

Fig. 2. Effects of carbon sources on mycelial growth and exopolysaccharide production in shake flask cultures of A. cylindracea ASI-9002: All

experimental data were mean ± SD of double determinations.

yM

ecail

oib l

mas

s)l/

g(

0

2

4

6

8

Ex

lo

po

ysira

hccad

/g( se

l)

0

1

2

3

4

Casei

npe

pton

e

Corn

stee

pliq

uor

Corn

stee

ppo

wde

r

Mar

tone

A-1

Mea

t pep

tone

Poly

pept

one

Soype

pton

eTr

yton

e

Yeast

extra

ct

Amm

oniu

mch

lorid

e

Amm

oniu

mci

trate

Amm

oniu

mni

trate

Amm

oniu

mph

osph

ate

Sodiu

mni

trate

Fig. 3. Effects of nitrogen sources on mycelial growth and exopolysaccharide production in shake flask cultures of A. cylindracea ASI-9002: All

experimental data were mean ± SD of double determinations.

1178 H.O. Kim et al. / Bioresource Technology 96 (2005) 1175–1182

achieved in the medium containing Martone A-1 (Fig.

3). In comparison with organic nitrogen sources, inor-

ganic nitrogen sources frequently yield relatively lower

mycelial growth and EPS production in liquid cultures

of mushrooms (Yang et al., 2003).

3.3. Effect of mineral source

The effect of mineral sources on mycelial growth and

EPS production was examined by employing various

mineral sources at a concentration of 7mM, where

two mineral ions, used in the basal medium, were re-

moved. Among the mineral sources examined, the myce-

lial growth and EPS production reached the highest

levels in the media containing CaCl2 and MgSO4 Æ7H2O(Fig. 4). These two bioelements have usually been recog-

nized as favorable for mycelial growth and EPS produc-

tion in liquid cultures of several basidiomycetes

(Chardonnet et al., 1999; Hwang et al., 2003; Okbaet al., 1998).

Chardonnet et al. (1999) investigated that external

Ca2+ can play an indirect role in fungal growth by alter-

yM

ceail

oib lm

sas

g( )l/

0

1

2

3

4

5

6

Exlopoy

/g( sedir ahccasl)

0

1

2

3

4

Co

lortn CaCl 2

SeFO 4

. H7 2O

K 2PHO 4 CK

l

KH 2OP

4

OSgM

4. H7 2O

Fig. 4. Effects of mineral sources on mycelial growth and exopolysaccharide production in shake flask cultures of A. cylindracea ASI-9002: All

experimental data were mean ± SD of double determinations. The control culture was carried out using basal medium without any mineral source.

H.O. Kim et al. / Bioresource Technology 96 (2005) 1175–1182 1179

ing internal Ca2+ that controls the cytoplasmic Ca2+

gradient, and the activity of fungal enzymes involved

in cell wall expansion. Direct effect of Ca2+ on the fungal

cell wall may also be a significant factor in cell mem-

brane permeability interactions. In contrast, Papagianni

(2004) suggested that Ca2+ accumulation seemed to inhi-bit the synthesis of fungal biopolymers, possibly through

an effect on enzymes such as b-glucan-synthases. Forhigher CaCl2 concentrations, the calcium ion content

of the cell wall increased, resulting in reduced protein

and neutral sugar contents. The effects of these agents

appear to be mediated by a tip-high gradient of cyto-

plasmic free Ca2+, which is obligatorily present and in-

volved in active growth. Mg2+ is essential to all fungi.It is a cofactor in enzymatic reactions, stabilizes the

plasma membrane, and its uptake is ATP dependent.

The positive action of Ca2+ and Mg2+ on mycelial

Table 3

Analysis of media on mycelial growth and exopolysaccharide production in s

projects

Mycelial biomass (g/l)

A B C D

K1 14.79 ± 0.95a 19.79 ± 1.46 19.46 ± 1.77 18.18 ± 1

K2 22.05 ± 1.39 19.79 ± 1.52 19.89 ± 1.51 21.08 ± 0

K3 24.67 ± 1.82b 21.93 ± 1.18 22.16 ± 0.88 22.25 ± 1

k1 4.93 ± 0.32 6.60 ± 0.49 6.49 ± 0.59 6.06 ± 0

k2 7.35 ± 0.46 6.60 ± 0.51 6.63 ± 0.50 7.03 ± 0

k3 8.22 ± 0.61 7.31 ± 0.39 7.39 ± 0.29 7.42 ± 0

R 3.29c ± 1.27 0.71 ± 1.20 0.90 ± 1.25 1.36 ± 1

Optimal level 3 3 3 3

a KAi ¼Pmycelial yield at Ai. Values are mean ± SD of double determin

b kAi ¼ kAi =3. Values are mean ± SD of double determinations.c RAi ¼ maxfkAi g �minfkAi g. Values are mean ± SD of double determina

growth and EPS production was obvious in the present

submerged cultures.

3.4. Optimization results by the orthogonal matrix method

The conventional �variation of one factor at a time�approach of optimization is not only time-consuming

but often incapable in its interactions. The method be-

comes impractical when a large number of components

in the medium have to be considered since too many

combinations have to be taken into account to optimize

the medium composition. To investigate the relation-

ships between variables of medium components and

optimize their concentrations for cell growth and meta-bolite production, the orthogonal matrix method can be

used. The orthogonal matrix method, as a result of the

suitable design of factors, can give effective responses.

hake flask cultures of Agrocybe cylindracea ASI-9002 with orthogonal

Exopolysaccharides (g/l)

A B C D

.42 4.26 ± 0.35 3.65 ± 0.43 4.43 ± 0.16 3.90 ± 0.35

.75 4.98 ± 0.59 5.08 ± 0.23 4.79 ± 0.38 4.00 ± 0.15

.99 4.63 ± 0.14 5.14 ± 0.42 4.65 ± 0.54 5.97 ± 0.58

.47 1.42 ± 0.12 1.22 ± 0.14 1.48 ± 0.05 1.30 ± 0.12

.25 1.66 ± 0.20 1.69 ± 0.08 1.60 ± 0.13 1.33 ± 0.05

.66 1.54 ± 0.05 1.71 ± 0.14 1.55 ± 0.18 1.99 ± 0.19

.26 0.24 ± 0.32 0.49 ± 0.35 0.12 ± 0.46 0.69 ± 0.33

2 3 2 3

ations.

tions.

Maltose (%)

0

2

4

6

8

104 6 8

Col 1 1 vs Ds DCW

Col 1 v 1 vs EPS S

Martone A-1 (%)0.2 0.4 0.6

0

1

2

3

4

ilecyM

oib la

/g( ssa

ml)

0

2

4

6

8

MgSO4. 7H2O (%)

0.04 0.09 0.14CaCl2 (%)

0.01 0.06 0.11

) l/g( se

dirahccasyl

op

oxE

0

1

2

3

Fig. 5. Intuitive analysis of the relationship between media, mycelial growth, and exopolysaccharide production in shake flask cultures of A.

cylindracea ASI-9002.

1180 H.O. Kim et al. / Bioresource Technology 96 (2005) 1175–1182

This implied that the selected conditions were the most

suitable in practice. In fact, they have been successfully

applied to the improvement of culture media for the

Time (d)0 2 4 6

yM

)l/g( ssa

moi

b lailec

0

2

4

6

8

10

12

14

yM

)l/g( ssa

moi

b lailec

0

2

4

6

8

10

12

14

16(A)

(B)

Fig. 6. Time profiles of mycelial growth and exopolysaccharide production

optimized medium (B). The data are result of one set of experiment.

production of primary and secondary metabolites on

fermentation process (Montgomery, 1999; Tarng et al.,

2002; Di et al., 2003).

8 10 12

ylo

pox

Es

)l/g( se

dirahcca

0

2

4

6

8

Hp

0

2

4

6

8

ylo

pox

Es

ccase

dirah

)l/g(

0

2

4

6

8H

p

0

2

4

6

8

in a 5-l stirred-tank fermenter under the basal medium (A) and the

H.O. Kim et al. / Bioresource Technology 96 (2005) 1175–1182 1181

During the optimization experiments, the fermenta-

tion conditions of temperature, initial pH, agitation

rate, and growth period were fixed at 28 �C, 6.0,150rpm, and 8days, respectively. The experimental re-

sults of the orthogonal design are shown in the last

two columns in Table 2. The effect of culture media onmycelial growth and EPS production was calculated

and the results are shown in Table 3. To obtain each fac-

tor, the intuitive analyses were performed as shown in

Fig. 5, based on statistical calculation using the data

in Table 3. In summary, the optimization results were

as follows: (1) to obtain high mycelial growth, the opti-

mized medium compositions were 80g/l maltose, 6g/l

Martone A-1, 1.4g/l MgSO4 Æ7H2O, and 1.1g/l CaCl2;(2) to obtain high EPS production, the optimized

medium compositions were 60g/l maltose, 6g/l Martone

A-1, 0.9g/l MgSO4 Æ7H2O, and 1.1g/l CaCl2.

3.5. Fermentation results

Fig. 6A shows the typical time courses of mycelial

growth and EPS production in a 5-l stirred-tank bioreac-tor. Under the basal culture condition (glucose 20g/l,

KH2PO4 0.46g/l, K2HPO4 1g/l, MgSO4 Æ7H2O 0.5g/l,

meat peptone 2g/l, yeast extract 2g/l, 25 �C and pH5.0),the maximum EPS production indicated 1.24g/l at 9days

of the fermentation while the mycelial growth reached

9.06g/l at 9days. Meanwhile, under the optimal culture

condition (maltose 60g/l, Martone A-1 6g/l, MgSO4 Æ7H2O 0.9g/l, CaCl2 1.1g/l, 25 �C and pH6.0), the maxi-mum EPS production indicated 3.0g/l at 10days of the

fermentation while mycelial growth reached 11.64g/l at

9days (Fig. 6B). The EPS concentration achieved at the

optimized culture condition was about three times higher

than that at the basal culture condition.

4. Conclusions

To date, many investigators have studied the produc-

tion of mushroom polysaccharides by submerged cul-

tures. Nevertheless, relatively few authors have used

statistical methodology for medium optimization in fer-

mentation process. The orthogonal matrix method was

proved to be a useful optimization technique for deter-

mining submerged culture condition of A. cylindracea.The optimization strategy established in this study may

be worth attempting with other mushroom fermentation

processes for enhanced production of mushroom poly-

saccharides, particularly those with industrial potential.

Acknowledgement

This work was supported by the RRC program of

MOST and KOSEF.

References

Bae, J.T., Sinha, J., Park, J.P., Song, C.H., Yun, J.W., 2000.

Optimization of submerged culture conditions for exo-biopolymer

production by Paecilimyces japonica. J. Microbiol. Biotechnol. 10,

482–487.

Chardonnet, C.O., Sams, C.E., Conway, W.S., 1999. Calcium effect on

the mycelial cell walls of Botrytis cinerea. Phytochemistry 52, 967–

973.

Chihara, G., Himuri, J., Maeda, Y.Y., Arai, Y., Fukuoka, F., 1970.

Fractionation and purification of the polysaccharides with marked

antitumor activity, especially, lentinan, from Lentinus edodes

(Berk) SING. Cancer Res. 30, 2776–2781.

Coello, N., Brito, L., Nonus, M., 2000. Biosynthesis LL-lysine by

Corynebacterium glutamicum grown on fish silage. Biores. Technol.

73, 221–225.

Di, X., Chan, K.K.C., Leung, H.W., Huie, C.W., 2003. Fingerprint

profiling of acid hydrolyzates of polysaccharides extracted from the

fruiting bodies and spores of Lingzhi by high-performance thin-

layer chromatography. J. Chromatogr. A 1018, 85–95.

Ergun, M., Mutlu, S.F., 2000. Application of a statistical technique to

the production of ethanol from sugar beet molasses by Saccharo-

myces cerevisiae. Biores. Technol. 73, 251–255.

Escamilla, S.E.M., Dendooven, L., Magana, I.P., Parra, S.R., Torre,

M.D., 2000. Optimization of gibberellic acid production by

immobilized Gibberella fujikuroi mycelium in fluidized bioreactor.

J. Biotechnol. 76, 147–155.

Gerlach, S.R., Siedenberg, D., Gerlach, D., Schtigerl, K., Giuseppin,

M.L.F., Hunik, J., 1998. Influence of reactor systems on the

morphology of Aspergillus awamori. Application of neural network

and cluster analysis for characterization of fungal morphology.

Process Biochem. 33, 601–615.

Ha, H.C., Park, S., Park, K.S., Lee, C.W., Jung, I.C., Kim, S.H.,

Kwon, Y.I., Lee, J.S., 1995. Isolation and purification of protein-

bound polysaccharides from the sawdust mycelia of Agrocybe

cylindracea. Kor. J. Mycol. 23, 121–128.

Hwang, H.J., Kim, S.W., Xu, C.P., Choi, J.W., Yun, J.W., 2003.

Production and molecular characteristics of four groups of

exopolysaccharides from submerge culture of Phellinus gilvus. J.

Appl. Microbiol. 94, 708–719.

Kiho, T., Yoshida, I., Nagai, K., Ukai, S., 1989. (1! 3)-a-DD-Glucanfrom an alkaline extract of Agrocybe cylindracea, and antitumor

activity of its o-carboxymethylated derivatives. Carbohydr. Res.

189, 273–279.

Kiho, T., Sobue, S., Ukai, S., 1994. Structural features and hypogly-

cemic activities of two polysaccharides from a hot-water extract of

Agrocybe cylindracea. Carbohydr. Res. 251, 81–87.

Kim, S.W., Hwang, H.J., Xu, C.P., Sung, J.M., Choi, J.W., Yun, J.W.,

2003. Optimization of submerged culture process for the produc-

tion of mycelial biomass and exo-polysaccharides by Cordyceps

militaris C738. J. Appl. Microbiol. 94, 120–126.

Kim, S.H., Jung, I.C., Kim, S., Y, Lee, J.S., Cho, H.J., Lee, H.W., Lee,

J.S., 1999. Characteristics and purification of polysaccharide

produced from Agrocybe cylindracea. Kor. J. Mycol. 27, 100–

106.

Kim, W.G., Lee, I.K., Kim, J.P., Ryoo, I.J., Koshino, H., Yoo, I.D.,

1997. New indole derivatives with free radical scavenging activity

from Agrocybe cylindracea. J. Nat. Prod. 60, 721–723.

Koshino, H., Lee, I.K., Kim, J.P., Kim, W.G., Uzawa, J., Yoo, I.D.,

1996. Agrocybenin, Novel class alkaloid from the Korean mush-

room Agrocybe cylindracea. Tetrahedron Lett. 37, 2550–4549.

Lee, I.K., Yun, B.S., Yoo, I.D., 1998. A nucleoside with lipid

peroxidation inhibitory activity from Agrocybe cylindracea. Kor.

J. Appl. Microbiol. Biotechnol. 26, 558–561.

Lee, J.H., Cho, S.M., Kim, H.M., Hong, N.D., Yoo, I.D., 1996.

Immunostimulating activity of polysaccharides from mycelia of

1182 H.O. Kim et al. / Bioresource Technology 96 (2005) 1175–1182

Phellinus linteus grown under different culture conditions. J.

Microbiol. Biotechnol. 6, 52–55.

Lee, J.S, Park, S., Park, G.S., 1989. Optimization of media compo-

sition and cultivation for the mycelial growth of Agrocybe

cylindracea. Kor. J. Food Sci. Technol. 21, 399–403.

Lee, M.T., Chen, W.C., Chou, C.C., 1997. Medium improvement by

orthogonal array designs for cholesterol oxidase production by

Rhodococcus equi No. 23. Process Biochem. 32, 697–703.

Li, Y., Chen, J., Lun, S.Y., Rui, X.S., 2001. Efficient pyruvate

production by a multi-vitamin auxotroph of Torulopsis glabrate:

key role and optimization of vitamin levels. Appl. Microbiol.

Biotechnol. 55, 680–685.

Maziero, R., Cavazzoni, V., Bononi, V.L.R., 1999. Screening of

basidiomycetes for the production of exopolysaccharide and

biomass in submerged culture. Rev. Microbiol. 30, 77–84.

Montgomery, D.C., 1999. Design and Analysis of Experiment, 4th ed.

Wiley, New York, pp. 627–631, 364–391.

Ngai, P.H.K., Wang, H.X., Ng, T.B., 2003. Purification and charac-

terization of a ubiquitin-like peptide with macrophage stimulating,

antiproliferative and ribonuclease activities from the mushroom

Agrocybe cylindracea. Peptides 24, 639–645.

Okba, A.K., Ogata, T., Matsubara, H., Matsuo, S., Doi, K., Ogata, S.,

1998. Effects of bacitracin and excess Mg2+ on submerged mycelial

growth of Streptomyces azureus. J. Ferment. Bioeng. 86, 28–33.

Papagianni, M., 2004. Fungal morphology and metabolite production

in submerged mycelial processes. Biotechnol. Adv. 22, 189–259.

Rosado, F.R., Germano, S., Carbonero, E.L., Casta, S.M.G.D.,

Iacomini, M., Kemmelmeier, C., 2003. Biomass and exopolysac-

charide production in submerged culture of Pleurotus ostreatoro-

seus SING and Pleurotus ostreatus ‘‘florida’’ (JACK.: FR.)

KUMMER. J. Basic. Microbiol. 43, 230–237.

Shu, C.H., Lung, M.Y., 2004. Effect of pH on the production

and molecular weight distribution of exopolysaccharide by

Antrodia camphorata in batch cultures. Process Biochem. 39,

931–937.

Song, C.H., Jeon, Y.J., Yang, B.K., Ra, K.S., Sung, J.M., 1998. The

anti-complementary activity of exo-polymers produced from

submerged mycelial cultures of higher fungi with particular

reference to Cordyceps militaris. J. Microbiol. Biotechnol. 8, 536–

539.

Tarng, Y.S., Juang, S.C., Chang, C.H., 2002. The use of grey-based

Taguchi methods to determine submerged arc welding process

parameters in hardfacing. J. Mater. Process. Technol. 128, 1–6.

Wang, H., Ng, T.B., Liu, Q., 2002. Isolation of a new heterodimeric

lectin with mitogenic activity from fruiting bodies of the mushroom

Agrocybe cylindracea. Life Sci. 70, 877–885.

Wang, Y., McNeil, B., 1995. pH effects on exopolysaccharide and

oxalic acid production in cultures of Sclerotium glucanicum.

Enzyme Microb. Technol. 17, 124–130.

Xu, C.P., Kim, S.W., Hwang, H.J., Choi, J.W., Yun, J.W., 2003.

Optimization of submerged culture conditions for mycelial growth

and exo-biopolymer production by Paecilomyces tenuipes C240.

Process Biochem. 38, 1025–1030.

Yang, F.C., Huang, H.C., Yang, M.J., 2003. The influence of

environmental conditions on the mycelial growth of Antrodia

cinnamomea in submerged cultures. Enzyme Microb. Technol. 33,

395–402.

Yoshida, I., Kiho, T., Usui, S., Sakushima, M., Ukai, S., 1996.

Polysaccharides in fungi. XXXV. Immunomodulating activities of

carboxymethylated derivatives of linear (1! 3)-a-DD-glucansextracted from the fruiting bodies of Agrocybe cylindracea and

Amanita muscaria. Biol. Pharm. Bull. 19, 114–121.