Phellinus sp.의 분리 및 균사체의 액체배양

강원도 양양군 일원 야산의 뽕나무에서 자생하고 있는 상황버섯 형태의 자실체를 채집하고, 그 자실체로부터 Phellnus속의 한 균주를 분리동정하였으며, 액체배양에 의하여 분리 균사체의 대량생산을 위한 배양조건, 배지조성 및 배양장치의 영향을 조사하였다. 자실체는 표면이 검으며 거칠고, 갓의 안쪽은 황갈색으로 평평하였으며, 자실층이 두터웠고(5-7 cm), 말굽형으로 조직이 매우 단단하여 전형적인 상황버섯의 형태를 나타내었다. 또, 조직 일부를 water agar에 이식하여 순수분리한 결과, 분리된 균사체는 황갈색으로, PDA 평판배지에서 자갈색의 색소를 분비하였으며, 균사는 clamp connection을 형성하지 않았고, 많은 branch가 있었다. 또 laccase, tyrosinase 및 peroxidase activity를 가지고 있어, 본 균주는 Phellinus속의 한 균주로 확인되었다. 플라스크 배양에서 균사체 생성을 위한 최적배양조건은 접종비 5%(v/v), 배지액량 70 mL, 교반속도 150rpm, 초기 pH 6, 배양온도 $30^{\circ}C$ 및 배양기간은 8일이었다. 또, 반응표면분석에 의한 최적 배지조성은 glucose 12.12 g/L, sucrose 12.12 g/L, yeast extract 11.15 g/L, malt extract 11.15 g/L, $KH_2PO_4$ 0.855 g/L 및 $CaCl_2$ 0.855 g/L 이었으며, 배양 4일 및 8일 후 균사체량은 각각 1.95 및 9.89 g/L 이었고, 최대 비생육속도 $0.020\;hr^{-1}$, 생산성은 1.25 g/L/day이었다. 그러나 서로 다른 3종의 배양장치를 사용한 결과, 3 L air-lift fermentor에서 통기량 2.5 vvm으로 4일간 배양하였을 때, 약 7.5g/L의 균사체를 얻을 수 있었으며, 이때, 최대 비증식속도는 $0.033\;hr^{-1}$, 생산성은 1.9g/L/day로, 같은 기간내 플라스크 배양시 보다 각각 1.7 및 4.2배가 향상된 결과를 얻을 수 있었다.

Fruit bodies similar to the Phellinus sp. residing on the mulberry were collected at Yang-yang in Kang-won-do province and one strain of Phellinus sp. was isolated from the fruit bodies. For mass production of the isolated mycelia in a submerged culture, the culture conditions, medium composition, and the effect of various culture systems on the mycelial growth, were investigated. The morphological characteristics of the fruit body were as follows: covered with blackish to black and rough, lower surface with yellowish-brown to dull-brown and smooth, 5-7 cm thick and hard woody. Also, the pure cultured mycelia showed yellowish-brown color, capability of purplish-brown pigment production on the PDA plate media, no-formation of clamp-connection, much binding branch, and enzyme activities such as laccase, tyrosinase and peroxidase. Therefore, pure cultured strain was identified to be Phellinus sp. In the flask culture, the optimum culture conditions for the mycelial production were obtained after cultivation of 8 days at inoculum level of 5%(v/v), media volume of 70 mL, 150 rpm, initial pH 6, and temperature of $30^{\circ}C$. Optimum medium composition from the response surface analysis were determined to be glucose 12.12 g/L, sucrose 12.12 g/L, yeast extract 11.15 g/L, malt extract 11.15 g/L, $KH_2PO_4$ 0.855 g/L and $CaCl_2$ 0.855 g/L. The production of the mycelia after 4 and 8 days of cultivation was 1.95 and 9.89 g/L, respectively. The maximum specific growth rate and productivity were $0.020\;hr^{-1}$ and 1.25 g/L/day, respectively. Among the three different culture systems for the growth of mycelia, the maximum mycelial dry weight of 7.5 g/L was obtained after cultivation of 4 days in the air-lift fermentor under aeration rate of 2.5 vvm. The maximum specific growth rate and productivity were $0.033\;hr^{-1}$ and 1.9 g/L/day, respectively, which were about 1.7 and 4.2 times higher than those of flask culture.