- 學位論文
橄欖油搭配金屬離子對牛樟菌生產三萜類的影響
Effect of Olive Oil and Metal Ion on the Production of Triterpenoid in Antrodia cinnamomea Culture
摘要
牛樟芝 ( Antrodia cinnamomea ) 於森林中生長遭到各種環境刺激:非生物性因子有日照、溫度、濕度等交替變化，以及源於生物體的生物性因子有牛樟樹 ( MJ、金屬離子 ) 、細菌 ( Peptidoglycan ) 、真菌 ( Chitosan ) 、昆蟲咬傷等誘發合成三萜類物質。若將牛樟菌絲體予以不同的環境刺激或添加外來物誘導，可能促使菌體專司生產三萜類的基因啟動，或幫助三萜合成路徑相關酵素合成，進一步提升三萜類總量產。 實驗以 MEM 為培養液，再加入1 % ( v/v ) 橄欖油 ( Olive oil；Oli ) 為碳源，嘗試比較茉莉酸甲酯 ( Methyl jasmonate；MJ)、幾丁聚醣 ( Chitosan；Chi ) 、銅離子 ( Cu2+ ) 等三種生物性因子進行三萜類生產研究。實驗發現銅離子被篩出有較佳的三萜類產量與比產量，分別為控制組的1.7和1.2倍。對六種金屬離子進行交互添加測試，結果發現1 % 橄欖油搭配亞鐵或錳離子不會抑制菌體生長，屬於不會激發三萜類比產量的類型，而搭配銅、鉀、鈣或鎂離子會抑制菌體生長，屬於會激發三萜類比產量的類型。於第0天同時添加100 μM 亞鐵離子與100 μM 鎂離子之後，三萜類產量為143.1 mg/L ( 控制組的2.5倍)。添加亞鐵與鎂金屬離子，造成菌體所含甾醇類組成比例改變；HPLC圖譜顯示同時添加100 μM 亞鐵離子與100 μM 鎂離子組，於第28天可促進62分鐘滯留峰的形成，每克菌乾重所含62分鐘峰分別為控制組的2.1、2.4倍。 實驗發現 MEA 固態培養添加1 % 橄欖油搭配 100 μM 亞鐵離子，再混合 10 μM 鎂離子，其菌乾重、三萜產量、三萜比產量、最大厚度與62分鐘峰含量，分別為控制組的1.8、2.6、1.5、3.0與3.9倍。初步實驗結果顯示特定金屬離子具有改善三萜類產量與比產量的效果，亞鐵離子不會抑制菌體生長亦可促進62分鐘峰的生成，未來可作為增加此成分之產量的有效方法。
並列摘要
Antrodia cinnamomea develops in forest and suffers environmental stimulating factors: abiotic factors - lighting, temperature and humidity and biotic factors from life - Cinnamomum Kanehirai Hay ( with MeJA and metal ions ) , bacteria ( with peptidoglycan ) , fungi ( with chitosan ) , insect bites etc., these may induce the synthesis of triterpenoids. If A. cinnamomea were to be provided by different environmental stimulating factors or additives, the total triterpenoid production might be further improved by promoting specific gene expression or activity of enzymes related to synthesized pathway. In this research, three biotic factors of methyl jasmonate ( MeJA ) , chitosan ( Chi ) , copper ion ( Cu2+ ) were used in a experiment based on malt extract medium ( MEM ) liquid culture added an extra carbon source of 1 % (v/v) olive oil, and results showed that addition of copper ion produce the more triterpenoid production and productivity, which were 1.7 and 1.2 times greater than control ( a pure MEM ) , respectively . An interactive addition test from six metal ions was carried out, 1 % olive oil coupled with ferrous or manganese ion didn't inhibite biomass production, these couldn't improve triterpenoid productivity, and 1 % olive oil coupled with copper, potassium, calcium, magnesium ion inhibited biomass production, these could improve triterpenoid productivity. The improved triterpenoid production of 143.1 mg/l ( 2.5 times ) was obtained at 100 μM Fe2+ and 100 μM Mg2+ added on 0th day. Addition of Fe2+ and Mg2+ caused changed proportion of composition of sterol for A. cinnamomea ; HPLC profiles showed that addition of Fe2+ and Mg2+ could promote the peak formation of 62 min of retention time per gram dry cell weight on 28th day, and it was 2.1 and 2.4 times greater than control group, respectively. In malt extract agar ( MEA ) solid culture, added 1% olive oil, 100 μM Fe2+ and 10 μM Mg2+, the dry cell weight, triterpenoid production, triterpenoid productivity, maximal thickness of colony and content of the peak of 62 min of retention time were 1.8, 2.6, 1.5, 3.0 and 3.9-fold of control obtained from a pure MEA culture. The initial experimental results demonstrated that a specific metal ion has the capibility of improved the triterpenoid production and productivity, and Fe2+ don't inhibit the biomass formation and can also promote the peak formation of 62 min of retention time. In the future, addition of ferrous ion might be a great method of increasing the production of the peak of 62 min of retention time.