Mycobiology 36(3) : 178-182 (2008)

© The Korean Society of Mycology

Submerged Culture of Phellinus linteus for Mass Production of Polysaccharides

June Woo Lee , Seong Jin Baek and Yong Seok Kim*
1 2

Department of Food Processing and Cooking, Kyungbuk College, Yeongjusi 750-712, Korea
1

Biotoxtech Co., Cheongwongun, Chungbuk, 363-883, Korea, Central Research Institute, Ilyang Pharm. Co. Ltd., Yonginsi 449-900, Korea
2

(Received September 5, 2008. Accepted September 19, 2008)

In order to increase the mycelial production of Phellinus linteus , which exhibits potent anticancer activity, some ingredients
of the medium used to culture P. linteus were investigated. The optimal medium composition for the production of Phellinus

linteus was determined to be as follows: fructose, 40 g/ ; yeast extract, 20 g/ ; K HPO , 0.46 g/ ; KH PO , 1.00 g/ ; MgSO ·7H O,
l l l l

0.50 g/ ; FeCl ·6 O, 0.01 g/ ; MnCl ·4H O, 0.036 g/ ; ZnCl , 0.03 g/ ; and CuSO ·7H O, 0.005 g/ . The optimal culture conditions
2 4 2 4 4 2

l l l l l

were determined to be as follows: temperature, 28 C; initial pH, 5.5; aeration, 0.6 vvm; and agitation, 100 rpm, respectively.
2 2 2 2 2 4 2
o

Under optimal composition and conditions, the maximum mycelial biomass achieved in a 5 jar fermentor was 29.9 g/ .
l l

KEYWORDS : Cultural condition, Optimal media, Phellinus linteus , Polysaccharide

Various mushrooms have been used in traditional medi- exploitation. In order to obtain polysaccharides from
cine for a long time. The higher Basidiomycetes have mushrooms, most investigators have focused their efforts
become a subject of great interest due to their diverse on cultivating mushrooms on solid artificial media (for
nutritional, medicinal, and pharmaceutical properties. Over fruiting body production) rather than submerged culture
the past three decades, many polysaccharides and polysac- (Brochers et al., 1999; Kues and Liu, 2000). Obviously
charide-protein complexes have been isolated from the submerged cultures give rise to the potential advantages
fruiting bodies and mycelia of mushrooms (Wasser, 2002). of higher mycelial production in a compact space and
Phellinus linteus, a yellow-orange colored mushroom shorter period of time without significant contamination
that grows well on mulberry trees, is a well-known fun- (Zhong and Tang, 2004; Hwang et al., 2003; Berovic et
gus in the genus Phellinus in the family Hymenochaeta- al., 2003). Therefore, in the present study, the submerged
ceae, and it has been used as an herb in traditional cultivation conditions were optimized for the production
medicine for many years in Asian countries (Kim et al., of polysaccharides from P. linteus.
2004). In traditional medicine, it has been known to have
curative effects on stomachaches, inflammation, tumors Materials and Methods

and so on. It is also used to improve overall health and

prevent various diseases, such as gastroenteric disorders, Mushroom. The fruiting body from P. linteus IY003
lymphatic disease and cancers (Cho et al., 2002). was previously reported to possess antitumor and antioxi-
In 1968 (Ikekawa et al., 1968), it was first reported that dant activities (Lee et al., 2006). To obtain a pure culture
the hot water extract from the fruiting body of Phellinus of P. linteus IY003, a single spore was isolated from the
linteus inhibited the growth of sarcoma 180 to about fruiting bodies of P. linteus IY003, placed in 2% water
96.7%. Since then, many investigators have documented a agar and incubated for 2 days at 28 C. The mycelia ger-
o

wide variety of further reports (Chi et al., 1996; Chung et minated from the single spore were transferred to potato
al., 1993; Kang et al., 1997; Han et al., 1999). The active dextrose agar (PDA) and incubated for 10 days at 28 C. o

polysaccharide from Phellinus linteus stimulates humoral

and cell-mediated immunity (Kim et al., 1996; Song et Inoculum preparation. P. linteus IY003 was initially
al., 1995). Acidic polysaccharides and proteoglycans from grown on PDA in a petri dish and then transferred to seed
P. linteus activate protein tyrosine kinase and protein culture medium. The seed culture was grown in a 500 ml
kinase C (Kim et al., 2003a, b). The ethanol extract flask containing 100 ml of medium at 28 C on a rotary
o

showed strong antiangiogenic and antioxidant activities shaker incubator at 120 rpm for 7 days.
(Song et al., 2003).
As P. linteus is very rare in nature, the amount of wild Culture condition. The flask culture experiments were
mushrooms available is not sufficient for commercial performed in a 500 ml flask containing 100 ml of media
after inoculation with 10% (v/v) of the seed culture. The
*Corresponding author <E-mail : kys0908@chol.com> fermentation medium was inoculated with 10% (v/v) of
178
 Culture of Phellinus linteus for Mass Production of Polysaccharides 179

the seed culture and then cultivated at 28 C in a 5 l Jar o

Table 2. Effects of various nitrogen sources on the mycelial
fermentor (KoBiotech Co., Korea). Unless otherwise spec- growth of Phellinus linteus IY003
ified, fermentations were performed under the following Nitrogen sources Dry mycelial weight (g/l)
conditions: temperature, 28 C; aeration rate, 0.6 vvm; agi-
o
Peptone 25.9
tation speed, 100 rpm; initial, pH 5.5; and working vol- Yeast extract 29.9
ume, 3.5 l. Malt extract 05.7
Tryptone 18.1
Analytical methods. The weight of the mycelium in the Soytone 10.2
culture medium was determined by filtration through pre- Casein 09.2
weighted dried Whatman filter paper No. 2 (Whatman Beef extract 15.1
International. UK) under suction, after washing the filter 3 Corn steep liquor 12.3
times with water, and drying to constant weight at 105 C o
NaNO 3 02.7
for 20 hrs. Residual fructose in the medium was assayed NH Cl4 01.0
by the DNS method. Viscosity measurements were per- P. linteus IY003 was cultured for 7 days at 28 C in medium con-
o

formed on samples collected from the fermentor at regu- taining 2% of the nitrogen source, 4% fructose, 0.036% K HPO , 2 4

lar intervals using a Brookfield RVTDV digital viscometer 0.1% KH PO , 0.05%, MgSO ·7H O, Mineral salt 1 ml, pH 5.5.
2 4 4 2

(Brookfield Engineering Laboratories, USA) fitted with a

small sample adapter. Antioxidant activity and nitric oxide to be the best carbon source for mycelial production. Con-
(NO) production were assayed as previously reported (Lee trary to our findings, other researchers have reported that
et al., 2006). mannose was the most suitable carbon source, and they
emphasized that different carbon sources slightly altered
Preparation of partially purified polysaccharides. the compositions of polysaccharides (Lee et al., 1995).
Culture broth of P. linteus IY003 was extracted 4 times Of the nitrogen sources tested, the best mycelial growth
with 2 volumes of distilled water at 121 C for 30 min. o
was observed in the medium containing yeast extract
After centrifugation (3,000 ×g), the supernatant was con- (Table 2). However, inorganic nitrogen sources, such as
centrated by evaporation at 75 C. Three volumes of etha-
o
sodium nitrate (NaNO ) and ammonium chloride (NH Cl),
3 4

nol were added to the supernatant, which was then stored were hardly utilized. In comparison with the results
at 4 C for 24 hours. After centrifugation, the precipitate
o
reported by other investigators, the nitrogen source
was ultrafiltrated (10 kDa cut-off) and freeze-dried. required for a liquid culture of P. linteus IY003 was dif-
ferent from that of P. linteus LI3202, i.e. mycelial growth
Results and Discussion was most favorable in medium containing peptone in
most P. linteus fermentation processes (Lee et al., 1995).
Effects of carbon and nitrogen sources. To find a suit- The optimum medium composition for P. linteus IY003
able carbon source, P. linteus IY003 was cultured in was determined from the results, as shown in Table 7.
media containing various carbon sources at a concentra-
tion of 4% (w/v) for 7 days. Of the 9 carbon sources Effects of pH and temperature. In order to investigate
examined, fructose, maltose, glucose and mannose were the effects of initial pH on mycelial growth, P. linteus
favorable to mycelial growth (Table 1). Fructose proved IY003 was cultivated in the optimized medium under dif-
ferent initial pHs (3.0~7.0) in a shake flask culture. Table
3 shows the effects of initial pH on the mycelial growth
Table 1. Effects of various carbon sources on the mycelial
growth of Phellinus linteus IY003
Carbon sources Dry mycelial weight (g/l) Table 3. Effects of initial pH on the mycelial growth of Phellinus
linteus IY003
Glucose 26.8
Galactose 09.6 Initial pH Dry mycelial weight (g/l)
Fructose 29.9 3.0 04.1
Mannose 25.8 4.0 13.9
Maltose 29.2 4.5 22.3
Sucrose 16.3 5.0 25.9
Lactose 15.4 5.5 29.9
Cellulose 18.3 6.0 28.0
Corn starch 19.1 7.0 18.6
P. linteus IY003 was cultured for 7 days at 28oC in medium con- P. linteus IY003 was cultured for 7 days at 28 C in the medium con-
o

taining 4% of the carbon source, 2% yeast extract, 0.036% K HPO , 2 4 taining 4% fructose, 1.5% yeast extract, 0.036% K HPO , 0.1%
2 4
0.1% KH PO , 0.05%, MgSO ·7H O, Mineral salt 1 ml, pH 5.5.
2 4 4 2 KH PO , 0.05%, MgSO ·7H O, Mineral salt 1 ml.
2 4 4 2
180 Lee et al.

Table 4. Effects of temperature on the mycelial growth of Table 6. Effects of agitation on the mycelial growth of Phellinus
Phellinus linteus IY003 linteus IY003
Temperature (oC) Dry mycelial weight (g/l) Agitation (rpm) Dry mycelial weight (g/l)
20 14.1 050 22.0
25 16.1 100 29.9
28 29.9 150 24.2
30 29.4 300 19.5
35 27.3 P. linteus IY003 was cultured for 7 days at 28 C by 0.6 vvm in o

P. linteus IY003 was cultured for 7 days in medium containing 4% medium containing 4% fructose, 1.5% yeast extract, 0.036% K HPO , 2 4

fructose, 1.5% yeast extract, 0.036% K HPO , 0.1% KH PO , 0.05%,

2 4 2 4
0.1% KH PO , 0.05%, MgSO ·7H O, Mineral salt 1 ml, pH 6.0.
2 4 4 2

MgSO ·7H O, Mineral salt 1 ml, pH 6.0.

4 2

Comparison to other media. Media typically used to

of P. linteus IY003. The highest mycelial concentration cultivate higher fungi (i.e., potato dextrose broth (PDB),
was obtained at a pH of 5.5. The optimum environmental mushroom complete medium (MCM) and Czapeck
conditions for the mycelial growth of Phellinus mush- medium) were compared to the optimized medium (Table
rooms in liquid cultures depend on the strain. Contrary to 8). Mycelial growth was best on the optimized medium,
our findings, the optimum pH for mycelial growth of P. followed by MCM, PDB, and Czapeck (Table 8), with
linteus L13202 was 7.0 (Lee et al., 1995). It has been maximum mycelium production rates of 29.9, 24.2, 18.6,
reported that the optimal pH for mycelial growth of a wide and 11.9 g/l, respectively. The initial pH of the fermenta-
variety of mushrooms is also acidic (Kim et al., 2002; Park tion broth of P. linteus IY003 slowly increased according
et al., 2001), and several P. linteus strains have an opti- to growth until it reached a maximum of 6.0 at the end of
mal pH of 5~7 (Choi and Lee, 2000; Kim et al., 2000). fermentation. However, the pH of the Czapeck media
To determine the optimal temperature for mycelial decreased.
growth, P. linteus IY003 was cultivated in shake flask cul-
ture at various temperatures (20~35 C), where the opti-
o
Results of fermentation in a Jar fermentor. Fig. 1
mum temperature was found to be 28 C (Table 4). It is
o
shows the typical time courses of mycelial growth and
comparable that many kinds of mushrooms have rela- fructose consumption in a 5 l jar fermentor. Under opti-
tively low optimal growth temperatures, ranging from 20
to 25 C in their submerged cultures.
o
Table 7. Optimized conditions for culture of Phellinus linteus
IY003
Effects of aeration and agitation. Mycelium produc- Factors Components Composition
tion by P. linteus IY003 was investigated with aeration
rates of 0.2, 0.4, 0.6, 0.8 and 1.0 vvm. The production of Fructose 40.0 (g/l)
Yeast extract 20.0 (g/l)
mycelia increased as the aeration rate increased. How- K HPO 0.46 (g/l)
ever, the optimum aeration rate was found to be 0.6 vvm, 2

KH PO
4

1.00 (g/l)
and mycelia production tended to decrease at an aeration
2 4

Media MgSO ·7H O 4 2 0.50 (g/l)

rate of 1.0 vvm (Table 5). FeCl ·6 O 2 2 0.01 (g/l)
To examine the effects of agitation speed on mycelial MnCl ·4H O 2 2 0.036 (g/l)
production, P. linteus IY003 was cultivated at 50, 100, ZnCl 2 0.03 (g/l)
150, and 300 rpm, under the standard conditions (28 C, o CuSO ·7H O 4 2 0.005 (g/l)
pH 5.5, 0.6 vvm). The optimum agitation speed was Temperature 28 Co

found to be 100 rpm (Table 6). pH 5.5

Conditions Time 7days
Aeration 0.6 vvm
Table 5. Effects of aeration on the mycelial growth of Phellinus Agitation 100 rpm
linteus IY003
Aeration (vvm) Dry mycelial weight (g/l) Table 8. Comparison of the mycelial growth of Phellinus linteus
0.2 16.0 IY003 in various media
0.4 17.7
0.6 29.9 Initial Final Dry mycelial
Media
0.8 27.2 pH pH weight (g/l)
1.0 22.8 Optimized media 5.5 6.01 29.9
P. linteus IY003 was cultured for 7 days at 28 C at 200 rpm in the
o Potato dextrose broth 5.5 5.84 18.6
medium containing 4% fructose, 1.5% yeast extract, 0.036% K HPO , MCM 5.5 5.99 24.2
Czapeck 5.5 5.21 11.9
2 4

0.1% KH PO , 0.05%, MgSO ·7H O, Mineral salt 1 ml, pH 6.0.

2 4 4 2
 Culture of Phellinus linteus for Mass Production of Polysaccharides 181

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