| Abstract: | 本實驗選用桑黃菌PL02（Phellinus linteus PL02）為試驗菌種，探討桑黃菌於三種不同固態基質（薏仁、黑豆及燕麥）培養發酵期間（30 °C /20天）之菌絲體生長情形，篩選出最適合桑黃菌生長之固態基質；再以篩選出之固態基質培養桑黃菌，並探討最適合桑黃菌菌絲體及多醣體生產之固態發酵培養條件，在最適固態培養條件下，針對桑黃固態發酵產物、未發酵基質及氣舉式發酵槽發酵液之熱水及乙醇萃取物進行生物活性（抑菌力、抗氧化能力及癌細胞存活率）之比較評估。研究結果顯示，最適合培養桑黃菌PL02之固態基質為燕麥，菌絲體產量在第12天時可達最高之494.11 mg/g。最適菌絲體及多醣體生成之固態培養條件為：培養溫度為30℃，水添加量為60%（v/w），接菌量為3ml，最適碳源為1% （w/w）之果糖，氮源為1% （w/w）之酵母萃取物，生長因子為0.3%（w/w）之葉酸、無機鹽類為0.1% （w/w）之氯化鈉，起始pH值6.0。以上述最適固態培養條件，可於培養第12天達到最高多醣體（61.73 mg/g）與菌絲體（705.30 mg/g）產量，分別增加為基礎培養基最高產量（432.30 mg/g及45.48 mg/g）之1.63及1.35倍。燕麥基質、桑黃燕麥及氣舉式發酵槽發酵液三者之多醣體分子量分佈以桑黃燕麥最為複雜，且桑黃燕麥多醣體分子量（4.54×104Da）為三者最高。β-(1→3)-D-glucan相對含量之測定亦以桑黃燕麥（29.09 μg/ml LE）最高。燕麥基質、桑黃燕麥及氣舉式發酵槽發酵液之熱水及乙醇萃取物抑菌力測定結果顯示在所使用之六種病原菌測定菌株（Bacillus cereus BCRC 10250, Escherichia coli BCRC 10239, Listeria monocytogene BCRC 14845, Pseudomonas aeruginosa BCRC 10261, Staphylococcus aureus BCRC 10451 and Salmonella typhimurium BCRC 10241）上並沒有任何抑菌效果。燕麥基質、桑黃燕麥及氣舉式發酵槽發酵液三者熱水及乙醇萃取物之抗氧化測試中，以桑黃燕麥熱水萃取物在DPPH自由基清除能力（IC50＝1.23 mg/ml）、螯合亞鐵離子能力（IC50＝5.26 mg/ml）、還原力（IC50＝1.18 mg/ml）及總抗氧化能力（IC50＝0.89 mg/ml）具有最佳之抗氧化活性。燕麥基質、桑黃燕麥及氣舉式發酵槽發酵液之熱水萃取物的抑制癌細胞能力皆優於乙醇萃取物。其中又以桑黃燕麥熱水萃取物的抑制效果最佳。經桑黃燕麥熱水萃取物處理（1000μg/ml，5% CO2/37℃/48hr）後，肺腺癌細胞（A-549）、乳癌細胞（MCF-7）及 肝癌細胞（HepG2）的存活率分別為46.29，55.75及63.33%，皆顯著（P<0.05）低於其他測試組。綜合上述結果，固態培養之桑黃燕麥較未發酵之燕麥基質及發酵液具更高的多醣體分子量及多醣體中β-(1→3)-D-glucan相對含量；樣品萃取物中，以熱水萃取物較乙醇萃取物具較高之生物活性，其中又以桑黃燕麥之熱水萃取物亦具最高之抗氧化力及抑制癌細胞生長能力等生物活性。
Three solid substrates (adlay, black bean, and oat) were used to study their effect on the mycelial growth of Phellinus linteus during solid-state fermentation (30°C/20days). The oat was chosen as the most suitable substrate to investigate the optimal solid- state culturing conditions for production of polysaccharide and mycelial biomass, and to compare the bioactivity（antibacterial, antioxidant and antitumor activities）of hot-water and ethanolic extracts from solid-state culture and submerged culture by this strain.Results showed that the optimal factors of solid-state culture were : incubation temperature of 30℃, water supplementation of 60% (v/w), inoculum volume of 3ml, fructose at 1% (w/w), yeast extract at 1% (w/w), folic acid at 0.3% (w/w), NaCl at 0.1% (w/w) and initial pH of 6.0. Under this optimized culturing condition, yields of polysaccharide (61.37 mg/g) and mycelial biomass（705.30 mg/g）obtained on the 12th day of incubation increased 1.63 and 1.35 times, respectively, as compared with those（45.48 mg/g and 432.30 mg/g, respectively）from basal medium.Higher molecular weight (4.54×104Da) and relative β-(1→3)-D-glucan content (29.09 μg/ml LE) of polysaccharide were observed from Phellinus-fermented oat, as compared with those from oat substrate and submerged culture using air lift fermentor.Both hot-water and ethanolic extracts from oats substrate, Phellinus-fermented oat and submerged culture demonstrated no antibacterial activities against the six bacterial strains (Bacillus cereus BCRC 10250, Escherichia coli BCRC 10239, Listeria monocytogene BCRC 14845, Pseudomonas aeruginosa BCRC 10261, Staphylococcus aureus BCRC 10451 and Salmonella typhimurium BCRC 10241) tested.The hot-water extract from Phellinus-fermented oat had the highest DPPH scavenging effect (IC50＝1.23 mg/ml), chelating ability on ferrous ion (IC50＝5.26 mg/ml) , reducing power (IC50＝1.18m g/ml) and total antioxidant capacity (IC50＝0.89 mg/ml) among all extracts.All hot-water extracts showed higher anti-tumor activity than ethanolic extracts, with the hot-water extract from Phellinus-fermented oat possessed the highest anti-tumor activity. After treated with hot-water extract from Phellinus-fermented oat (1000μg/ml, 5% CO2/37℃/48hr), viabilities of lung-cancer cells (A-549), breast-cancer cells (MCF-7) and liver-cancer cells (HepG2) were 46.29%, 55.75% and 63.33%, respectively, and were significantly (P<0.05) lower than those of all other extracts.In conclusion, Phellinus-fermented oat possessed polysaccharide with higher molecular weight and relative β-(1→3)-D-glucan content than those of submerged culture and unfermented oat substrate. Extracts from Phellinus-fermented oat showed better bioactivity than those from submerged culture. Among the extracts, hot-water extract had higher bioactivity than ethanolic extracts. |
