Culture Conditions for Mycelial Growth of Coriolus versicolor.

Coriolus versicolor, is one of the most popular medicinal mushrooms due its various biologically active components. This study was conducted to obtain basic information regarding the mycelial culture conditions of C. versicolor. Based on the culture, and MCM media were suitable for the mycelial growth of the mushroom. The optimum carbon and nitrogen sources were dextrin and yeast extract, respectively, and the optimum C/N ratio was 10 to 2 when 2% glucose was used. Other minor components required for optimal growth included thiamine-HCl and biotin as vitamins, succinic acid, lactic acid and citric acid as organic acids, as well as MgSO4·7H2O as mineral salts.

It is estimated that there are 140,000 species of mushrooms worldwide, yet only 10% have been identified to date [1]. n class="Species">Mushrooms have long been valued as edible and medicinal resources. Coriolus versicolor, which belongs to polyporaceae of basidiomycetes, is a wood-rotting fungi and can be easily found in the natural environment. It has been reported that around 10 species of C. versicolor grow naturally in Korea as well [2]. Morphologically, C. versicolor has a thin, solid and oval pileus, and it is characterized as an annual mushroom living in stock of old needleleaf trees or broadleaf trees. Locally, C. versicolor is called 'Ungi' and is often used as a home remedy or a dietary supplement.

C. versicolor reportedly contains a variety of enzymes, including lignin peroxidase, manganese peroxidase and laccase [3-5].

The mushroom reportedly contains a variety of biologically active components, including bitter n class="Chemical">triterpenoids, alnusenone, friedelin, α-D-glucan and β-D-glucan [6-9]. Especially, C. versicolor has been actively researched, since it is known to have many pharmacological effects. Ever since Tsukagoshi and Ophashi [10] found that protein bound-polysaccharide has anti-tumor activity against sarcoma-180, the effort to apply to wider industry has continued. This study was conducted to determine the culture conditions for the optimal mycelial growth of C. versicolor.

Materials and Methods

Fungal isolates

The isolates of C. versicolor used in this study are listed in Table 1. C. versicolor ASI 16003, ASI 16006, ASI 16008, C. pubescens ASI 16002 and C. brevis ASI 16007 were obtained from the Rural Development Administration of Korea. C. versicolor GBCV-01 was collected in the wild. All isolates were maintained on potato dextrose agar (PDA).

Table 1

List of Coriolus versicolor strains used in this study

Effect of pH

To determine the optimal pH value for growth of C. versicolor, 5 diameter plugs were removed from 5-day-old cultures of C. versicolor grown on PDA using a cork borer. The plugs were then placed on the center of PDA plates with an adjusted pH range from 4 to 9 using 1 N NaOH or HCl. Samples were then incubated in the dark for 4 days at 25℃. The mycelial growth was measured according to the method described by Shim et al. [11].

Temperature

Growth of the mushrooms was evaluated at temperatures ranging from 10~35℃. The fungi were cultured on PDA for 5 days, and mycelial growth was determined as described above.

Culture media

Twelve different culture media were screened to determine the optimal medium for the mycelial growth of C. versicolor (Table 2). All media were sterilized for 20 min at 121℃ and then aseptically poured into plastic petri dishes. Inoculum was then removed from 5-day-old cultures of n class="Species">C. versicolor grown on PDA at 25℃, after which a mycelial disk (5 in diameter) was placed in the center of the prepared media. The fungi were then incubated in the dark for 4 days at 25℃, after which the mycelial growth and density of the colonies were examined.

Table 2

Composition of media used in this study

PDA, potato dextrose agar; MEA, malt extract agar; YEA, yeast extract agar; MCM, mushroom complete medium.

Effect of favorable nutrient sources

Carbon sources

Suitable carbon sources were screened by culturing the n class="Species">mushroom on mushroom minimal media (MMM; 20 g of dextrose, 0.5 g of MgSO4, 0.46 g of KH2PO4, 1 g of K2HPO4, 2 g of asparagine, 120 µg of thiamine-HCl, 20 g of agar, 1,000 mL of distilled water [DW]) supplemented with one-tenth carbon sources at a concentration of 2%. The fungi were incubated in the dark for 5 days at 25℃, after which the mycelial growth and density of the colonies were evaluated.

Nitrogen sources

To determine the optimal nitrogen source for the mycelial growth of C. versicolor, mushrooms were cultured on MMM supplemented with one of 12 nitrogen sources, each at a concentration of 0.2%. A 5 diameter plug of C. versicolor was placed in the center of the petri dish, which was incubated in the dark for 5 days at 25℃. The mycelial growth and density of the colonies were then examined.

C/N ratio

To determine the optimal C/N n class="Species">ratio, MMM were prepared using 10, 8, 6, 4, 2, 1, 0.4 and 0.2% glucose as the carbon source and 0.2% NaNO3 as the nitrogen source, giving C/N ratios of 50 : 1, 40 : 1, 30 : 1, 20 : 1, 10 : 1, 5 : 1, 2 : 1 and 1 : 1, respectively. The petri dishes were then inoculated with C. versicolor and incubated in the dark for 6 days at 25℃, after which the mycelial growth and density of the colonies were examined.

Vitamins

To determine which vitamins are suitable for the mycelial growth of C. versicolor, n class="Species">mushrooms were cultured on sterilized MMM that had been amended with thiamine-HCl (0.1 mg/L), riboflavin (0.5 mg/L), biotin (0.005mg/L), pyridoxine (0.5mg/L) or nicotinamide (2.0mg/L), followed by filtration through a metrical membrane filter with a pore size of 0.2 µm. The petri dishes were incubated in the dark for 5 days at 25℃, after which the mycelial growth and density of the colonies were examined.

Organic acid

To screen for mineral salts suitable for the mycelial growth of n class="Species">C. versicolor, MMM was prepared using acetic acid, citric acid, maleic acid, lactic acid, succinic acid or fumaric acid at a concentration of 0.1%. The petri dishes were then inoculated with C. versicolor and cultured in the dark for 5 days at 25℃, after which the mycelial growth and density of the colonies were examined.

Mineral salt

To screen for mineral salts suitable for the mycelial growth of n class="Species">C. versicolor, mushrooms were cultured on YM solid media (5 g of peptone, 3 g of yeast extract, 3 g of malt extract, 10 g of dextrose, 20 g of agar and 1,000 mL of DW. Consider specifying, also specify if Millipore water supplemented with one-ninth mineral salts at a concentration of 0.1%. The petri dishes were inoculated with C. versicolor and cultured in the dark for 5 days at 25℃. After which the mycelial growth and density of the colonies were examined.

Results and Discussion

Favorable mycelial growth of C. versicolor was obtained within the pH range of 4~6. Among the six strains, n class="Species">C. pubescens isolate ASI 16002 showed the biggest colony with a diameter of 76.7 mm at pH 4 (Table 3). The optimum pH range for the growth of C. versicolor has been reported to be 5.0~5.8 [12]. The results of the present study suggest that the growth of C. versicolor mycelia can occurwithin a specific pH range.

Table 3

Effect of pH on the mycelial growth of Coriolus versicolor at 25℃

SC, somewhat compact; T, thin; ST, somewhat thin.

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (p < 0.05). Results shown are the mean ± SD of three replicates.

Effect of temperature

Temperatures ranging from 25~30℃ were found to be suitable for the mycelial growth of n class="Species">C. versicolor (Fig. 1). However, the mycelial growth of C. versicolor was suppressed rapidly at temperatures above 30℃ and below 20℃. These findings are in agreement with the results of a study conducted by Park et al. [12], who reported that the optimum temperature for the growth of C. versicolor was 25~30℃.

Fig. 1

Mycelial growth of Coriolus versicolor on for 5 days at different temperatures. Vertical bars show the standard errors (n = 3).

Screening for suitable culture media

The mycelial growth of C. versicolor was favorable in n class="Chemical">PDA, MEA and malt yeast extract, whereas it was poor in Czapek Dox, glucose peptone and Hennerberg (Table 4). The mycelial growth of C. versicolor isolate ASI 16003 was lower than the other strains. The mycelial densities of C. versicolor were favorable in MEA but poor in Czapek Dox, Leonian, Hennerberg, Lily and Hoppkins. Shim et al. [13] also reported that PDA, YMA, mushroom complete medium and Hamada were suitable for the growth of Macrolepiota procera, whereas Czapex Dox and glucose peptone media were not.

Table 4

Effect of culture medium on the mycelial growth of Coriolus versicolor at 25℃

PDA, potato dextrose agar; MEA, malt extract agar; YEA, yeast extract agar; MCM, mushroom complete medium; ST, somewhat thin; C, compact; SC, somewhat compact; T, thin.

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (p < 0.05). Results shown are the mean ± SD of three replicates.

Dextrin, fructose and mannose were found to promote the mycelial growth of C. versicolor (Table 5). Of the 10 carbon sources evaluated, mannose led to the formation of C. pubescens isolate ASI 16002 colonies with the largest diameter (72.5 mm). The mycelial density of C. versicolor isolate ASI 16003 was somewhat compact for all carbon sources. Jeong et al. [14] reported that the optimum carbon source for the growth of G. applanatum is glucose while Jayasinghe et al. [15] reported that dextrin is the best carbon source for the mycelial growth of G. lucidum. Griffin [16] suggested that mannose and fructose are the most commonly utilized sugars after glucose.

Table 5

Effect of carbon source on the mycelial growth of Coriolus versicolor at 25℃

SC, somewhat compact; ST, somewhat thin; T, thin.

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (p < 0.05). Results shown are the mean ± SD of three replicates.

The nitrogen sources that promoted the best mycelial growth of C. versicolor were yeast extract and malt extract (Table 6). The mycelial densities of all C. versicolor strains were compact when grown in the presence of yeast extract. Among the 13 nitrogen sources evaluated, yeast extract resulted in the formation of C. pubescens isolate ASI 16002 colonies with a diameter of 77 mm. Jeong et al. [14] reported that the optimum nitrogen source for the culture of G. applanatum is corn steep powder (10%).

Table 6

Effect of nitrogen source on the mycelial growth of Coriolus versicolor at 25℃

C, compact; ST, somewhat thin; SC, somewhat compact; T, thin.

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (p < 0.05). Results shown are the mean ± SD of three replicates.

C/N ratios

The C/N ratios that promoted the mycelial growth of C. versicolor were 2 : 1, 5 : 1 and 10 : 1 (Table 7, Fig. 2). Generally, the mycelial density of C. versicolor is thin for all C/N ratios. Among the eight C/N ratios evaluated, a C/N ratio of 10 : 1 resulted in the growth of C. pubescens isolate ASI 16002 colonies with a diameter of 84.0 mm. Jo et al. [17] reported that the optimum C/N ratios for culture of Phellinus spp. are 10 : 1 and 5 : 1.

Table 7

Effect of C/N ratio on the mycelial growth of Coriolus versicolor at 25℃

SC, somewhat compact; ST, somewhat thin; T, thin.

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (p < 0.05). Results shown are the mean ± SD of three replicates.

Fig. 2

Mycelial growth of Coriolus versicolor isolate ASI 16008 on different C/N ratios. A, 50; B, 40; C, 30; D, 20; E, 10; F, 5; G, 2; H, 1.

In order to evaluate the effect of vitamins, five varieties of vitamins were added to the MMM medium. The results show that thiamine-HCl produced excellent growth of C. versicolor mycelia (Table 8). After 5 days of cultivation, the diameter of the C. pubescens isolate ASI 16002 colonies grown in thiamine-HCl and nicotinamide were 68.0 mm and 63.7 mm, respectively. Cho et al. [18] reported that the optimum culture vitamins of G. lucidum are nicotinic acid and pantothenic acid.

Table 8

Effect of vitamins on the mycelial growth of Coriolus versicolor at 25℃

SC, somewhat compact; ST, somewhat thin; T, thin.

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (p < 0.05). Results shown are the mean ± SD of three replicates.

Organic acids

Of the various organic acids were added to the MMM medium, succinic acid, citric acid and lactic acid were found to be excellent for the mycelial growth of C. versicolor (Table 9). After 5 days of cultivation, the diameter of C. pubescens isolate ASI 16002 colonies grown in the presence of citric acid and lactic acid were 66.0 mm and 63.7 mm, respectively.

Table 9

Effect of organic acids on the mycelial growth of Coriolus versicolor at 25℃

ST, somewhat thin; T, thin; SC, somewhat compact.

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (p < 0.05). Results shown are the mean ± SD of three replicates.

Mineral salts

To evaluate the effect of various mineral salts on the mycelial growth of C. versicolor, 8 types of mineral salts were added to YM medium. MgSO4·7H2O, KCl, and KH2PO4 were found to be excellent for the mycelial growth of C. versicolor, whereas ZnSO4·7H2O resulted in mostly negative growth (Table 10). Chi et al. [19] reported that the optimum growth of Phellinus linteus occurs when MgSO4·7H2O is used as the mineral salt.

Table 10

Effect of mineral salts on the mycelial growth of Coriolus versicolor at 25℃

SC, somewhat compact; ST, somewhat thin; T, thin.

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (p < 0.05). Results shown are the mean ± SD of three replicates.