Submerged Culture of Phellinus linteus for Mass Production of Polysaccharides.

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/l; yeast extract, 20 g/l; K2HPO4, 0.46 g/l; KH2PO4, 1.00 g/l; MgSO4·7H2O, 0.50 g/l; FeCl2·62O, 0.01 g/l; MnCl2·4H2O, 0.036 g/l; ZnCl2, 0.03 g/l; and CuSO4·7H2O, 0.005 g/l. The optimal culture conditions were determined to be as follows: temperature, 28℃; initial pH, 5.5; aeration, 0.6 vvm; and agitation, 100 rpm, respectively. Under optimal composition and conditions, the maximum mycelial biomass achieved in a 5 l jar fermentor was 29.9 g/l.

Various mushrooms have been used in traditional medicine for a long time. The higher Basidiomycetes have become a subject of great interest due to their diverse nutritional, medicinal, and pharmaceutical properties. Over the past three decades, many polysaccharides and polysaccharide-protein complexes have been isolated from the fruiting bodies and mycelia of mushrooms (Wasser, 2002).

Phellinus linteus, a yellow-orange colored mushroom that grows well on mulberry trees, is a well-known fungus in the genus Phellinus in the family Hymenochaetaceae, and it has been used as an herb in traditional medicine for many years in Asian countries (Kim et al., 2004). In traditional medicine, it has been known to have curative effects on stomachaches, inflammation, tumors and so on. It is also used to improve overall health and prevent various diseases, such as gastroenteric disorders, lymphatic disease and cancers (Cho et al., 2002).

In 1968 (Ikekawa et al., 1968), it was first reported that the hot water extract from the fruiting body of Phellinus linteus inhibited the growth of sarcoma 180 to about 96.7%. Since then, many investigators have documented a wide variety of further reports (Chi et al., 1996; Chung et al., 1993; Kang et al., 1997; Han et al., 1999). The active polysaccharide from Phellinus linteus stimulates humoral and cell-mediated immunity (Kim et al., 1996; Song et al., 1995). Acidic polysaccharides and proteoglycans from P. linteus activate protein tyrosine kinase and protein kinase C (Kim et al., 2003a, b). The ethanol extract showed strong antiangiogenic and antioxidant activities (Song et al., 2003).

As P. linteus is very rare in nature, the amount of wild mushrooms available is not sufficient for commercial exploitation. In order to obtain polysaccharides from mushrooms, most investigators have focused their efforts on cultivating mushrooms on solid artificial media (for fruiting body production) rather than submerged culture (Brochers et al., 1999; Kues and Liu, 2000). Obviously submerged cultures give rise to the potential advantages of higher mycelial production in a compact space and shorter period of time without significant contamination (Zhong and Tang, 2004; Hwang et al., 2003; Berovic et al., 2003). Therefore, in the present study, the submerged cultivation conditions were optimized for the production of polysaccharides from P. linteus.

Materials and Methods

Mushroom

The fruiting body from P. linteus IY003 was previously reported to possess antitumor and antioxidant activities (Lee et al., 2006). To obtain a pure culture of P. linteus IY003, a single spore was isolated from the fruiting bodies of P. linteus IY003, placed in 2% water agar and incubated for 2 days at 28℃. The mycelia germinated from the single spore were transferred to potato dextrose agar (PDA) and incubated for 10 days at 28℃.

Inoculum preparation

P. linteus IY003 was initially grown on PDA in a petri dish and then transferred to seed culture medium. The seed culture was grown in a 500 ml flask containing 100 ml of medium at 28℃ on a rotary shaker incubator at 120 rpm for 7 days.

Culture condition

The flask culture experiments were performed in a 500 ml flask containing 100 ml of media after inoculation with 10% (v/v) of the seed culture. The fermentation medium was inoculated with 10% (v/v) of the seed culture and then cultivated at 28℃ in a 5 l Jar fermentor (KoBiotech Co., Korea). Unless otherwise specified, fermentations were performed under the following conditions: temperature, 28℃; aeration rate, 0.6 vvm; agitation speed, 100 rpm; initial, pH 5.5; and working volume, 3.5 l.

Analytical methods

The weight of the mycelium in the culture medium was determined by filtration through pre-weighted dried Whatman filter paper No. 2 (Whatman International. UK) under suction, after washing the filter 3 times with water, and drying to constant weight at 105℃ for 20 hrs. Residual fructose in the medium was assayed by the DNS method. Viscosity measurements were performed on samples collected from the fermentor at regular intervals using a Brookfield RVTDV digital viscometer (Brookfield Engineering Laboratories, USA) fitted with a small sample adapter. Antioxidant activity and nitric oxide (NO) production were assayed as previously reported (Lee et al., 2006).

Preparation of partially purified polysaccharides

Culture broth of P. linteus IY003 was extracted 4 times with 2 volumes of distilled water at 121℃ for 30 min. After centrifugation (3,000 ×g), the supernatant was concentrated by evaporation at 75℃. Three volumes of ethanol were added to the supernatant, which was then stored at 4℃ for 24 hours. After centrifugation, the precipitate was ultrafiltrated (10 kDa cut-off) and freeze-dried.

Results and Discussion

Effects of carbon and nitrogen sources

To find a suitable carbon source, P. linteus IY003 was cultured in media containing various carbon sources at a concentration of 4% (w/v) for 7 days. Of the 9 carbon sources examined, fructose, maltose, glucose and mannose were favorable to mycelial growth (Table 1). Fructose proved to be the best carbon source for mycelial production. Contrary to our findings, other researchers have reported that mannose was the most suitable carbon source, and they emphasized that different carbon sources slightly altered the compositions of polysaccharides (Lee et al., 1995).

Table 1

Effects of various carbon sources on the mycelial growth of Phellinus linteus IY003

P. linteus IY003 was cultured for 7 days at 28℃ in medium containing 4% of the carbon source, 2% yeast extract, 0.036% K2HPO4, 0.1% KH2PO4, 0.05%, MgSO4·7H2O, Mineral salt 1 ml, pH 5.5.

Of the nitrogen sources tested, the best mycelial growth was observed in the medium containing yeast extract (Table 2). However, inorganic nitrogen sources, such as sodium nitrate (NaNO3) and ammonium chloride (NH4Cl), were hardly utilized. In comparison with the results reported by other investigators, the nitrogen source required for a liquid culture of P. linteus IY003 was different from that of P. linteus LI3202, i.e. mycelial growth was most favorable in medium containing peptone in most P. linteus fermentation processes (Lee et al., 1995). The optimum medium composition for P. linteus IY003 was determined from the results, as shown in Table 7.

Table 2

Effects of various nitrogen sources on the mycelial growth of Phellinus linteus IY003

P. linteus IY003 was cultured for 7 days at 28℃ in medium containing 2% of the nitrogen source, 4% fructose, 0.036% K2HPO4, 0.1% KH2PO4, 0.05%, MgSO4·7H2O, Mineral salt 1 ml, pH 5.5.

Table 7

Optimized conditions for culture of Phellinus linteus IY003

Effects of pH and temperature

In order to investigate the effects of initial pH on mycelial growth, P. linteus IY003 was cultivated in the optimized medium under different initial pHs (3.0~7.0) in a shake flask culture. Table 3 shows the effects of initial pH on the mycelial growth of P. linteus IY003. The highest mycelial concentration was obtained at a pH of 5.5. The optimum environmental conditions for the mycelial growth of Phellinus mushrooms in liquid cultures depend on the strain. Contrary to our findings, the optimum pH for mycelial growth of P. linteus L13202 was 7.0 (Lee et al., 1995). It has been reported that the optimal pH for mycelial growth of a wide variety of mushrooms is also acidic (Kim et al., 2002; Park et al., 2001), and several P. linteus strains have an optimal pH of 5~7 (Choi and Lee, 2000; Kim et al., 2000).

Table 3

Effects of initial pH on the mycelial growth of Phellinus linteus IY003

P. linteus IY003 was cultured for 7 days at 28℃ in the medium containing 4% fructose, 1.5% yeast extract, 0.036% K2HPO4, 0.1% KH2PO4, 0.05%, MgSO4·7H2O, Mineral salt 1 ml.

To determine the optimal temperature for mycelial growth, P. linteus IY003 was cultivated in shake flask culture at various temperatures (20~35℃), where the optimum temperature was found to be 28℃ (Table 4). It is comparable that many kinds of mushrooms have relatively low optimal growth temperatures, ranging from 20 to 25℃ in their submerged cultures.

Table 4

Effects of temperature on the mycelial growth of Phellinus linteus IY003

P. linteus IY003 was cultured for 7 days in medium containing 4% fructose, 1.5% yeast extract, 0.036% K2HPO4, 0.1% KH2PO4, 0.05%, MgSO4·7H2O, Mineral salt 1 ml, pH 6.0.

Effects of aeration and agitation

Mycelium production 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 mycelia increased as the aeration rate increased. However, the optimum aeration rate was found to be 0.6 vvm, and mycelia production tended to decrease at an aeration rate of 1.0 vvm (Table 5).

Table 5

Effects of aeration on the mycelial growth of Phellinus linteus IY003

P. linteus IY003 was cultured for 7 days at 28℃ at 200 rpm in the medium containing 4% fructose, 1.5% yeast extract, 0.036% K2HPO4, 0.1% KH2PO4, 0.05%, MgSO4·7H2O, Mineral salt 1 ml, pH 6.0.

To examine the effects of agitation speed on mycelial production, P. linteus IY003 was cultivated at 50, 100, 150, and 300 rpm, under the standard conditions (28℃, pH 5.5, 0.6 vvm). The optimum agitation speed was found to be 100 rpm (Table 6).

Table 6

Effects of agitation on the mycelial growth of Phellinus linteus IY003

P. linteus IY003 was cultured for 7 days at 28℃ by 0.6 vvm in medium containing 4% fructose, 1.5% yeast extract, 0.036% K2HPO4, 0.1% KH2PO4, 0.05%, MgSO4·7H2O, Mineral salt 1 ml, pH 6.0.

Comparison to other media

Media typically used to cultivate higher fungi (i.e., potato dextrose broth (PDB), mushroom complete medium (MCM) and Czapeck medium) were compared to the optimized medium (Table 8). Mycelial growth was best on the optimized medium, followed by MCM, PDB, and Czapeck (Table 8), with maximum mycelium production rates of 29.9, 24.2, 18.6, and 11.9 g/l, respectively. The initial pH of the fermentation broth of P. linteus IY003 slowly increased according to growth until it reached a maximum of 6.0 at the end of fermentation. However, the pH of the Czapeck media decreased.

Table 8

Comparison of the mycelial growth of Phellinus linteus IY003 in various media

Results of fermentation in a Jar fermentor

Fig. 1 shows the typical time courses of mycelial growth and fructose consumption in a 5 l jar fermentor. Under optimum culture conditions, the maximum mycelial production rate was 29.9 g/l at 144 h of fermentation. To the best of our knowledge, the mycelial production obtained in this study is the highest yield amongst liquid cultures of P. linteus reported in the literature. The apparent viscosity of the fermentation broth of P. linteus IY003 increased rapidly during the exponential phase, but a rapid decrease was detected in a later stage. This drop in viscosity may be attributable to the occurrence of severe fragmentation in mycelial clumps of P. linteus IY003. Based on the results reported by another investigator, the apparent viscosity was influenced by mycelial fragmentation (Hwang et al., 2004).

Fig. 1

Mycelial growth and fructose consumption from Phellinus linteus in a 5 l fermentor. Mycelial weight (•), Residual fructose (○), Viscosity (▪), pH (□).

Yield and characterization of polysaccharides

The dry weight of polysaccharides extracted by hot water from the culture broth of P. linteus IY003 was 5.4 g. This extract showed high antioxidant activities (72.86%) by inhibiting lipid peroxidation in rat liver homogenate treated with 1mg/ml of the extract. Moreover, the production of nitric oxide (NO) in RAW264.7 (RAW) cells was 28.1 ± 1.8 nM (10 mg/ml treatment). This result indicated that hot water-extracted polysaccharides from the culture broth of P. linteus IY003 may have immunomodulating and antioxidant activities.