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標題: 應用義大利白松露內生菌與灰白松露生產胞外多醣體和總三萜之研究與其代謝物之鑑定
Study on Exopolysaccharide and Triterpenoid Production from a Truffle Endophytic Fungus Hypocreales sp. NCHU01
作者: Chiung-Wen Yeh
關鍵字: Tuber magnatum
Tuber borchii
endophytic fungus
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摘要: 近年來,廣泛地利用液態發酵培養食用菇類生產菌體和其代謝產物,已經成為食用菇類的一種趨勢。而食用菇類最吸引人的地方,在於它的代謝產物能夠抗腫瘤活性、保肝作用等多種醫療作用。 本論文主要包含三個部分,第一部分是以義大利白松露內生菌(Hypocreales sp. NCHU01)開發液態醱酵生產菌絲體和多醣體,利用回應曲面設計法,尋找最適化培養基生產多醣體,碳源、氮源、溫度、pH、鎂離子及鉀離子為設計因子,利用回應曲面設計法來找出最佳的培養基組成,結果顯示以蔗糖 10.60 g/L、yeast extract 10.92 g/L、MgSO4 1.0 g/L、KH2PO4 1.01 g/L,培養基最適初始pH和醱酵溫度,分別為6.5和25.0℃在恆溫震盪箱以100 rpm的操作條件下,可得到最佳的胞外多醣體 1329.25 mg/L,胞外多醣體為未最佳化前的3.34倍 第二部分研究以義大利白松露內生菌開發液態醱酵生產總三萜,研究結果,在碳源測試實驗中,當以sucrose濃度為10 g/L時,可得到較高的總三萜量132.26±1.77 mg/L;在氮源測試實驗中,當以yeast extract 濃度為15 g/L時,可得到較高的總三萜量157.68±14.33 mg/L。為了提升總三萜產量,添加不同的濃度的亞麻油酸、棕櫚酸、硬脂酸、幾丁聚醣、氯化鈣和樟木萃取液,結果顯示,當以2 g/L亞麻油酸和125 mg/L幾丁聚醣產總三萜量為最多,分別為327.49±42.15 mg/L及343.16±9.25 mg/L。最後以松露子實體與內生菌菌絲體進行氣相層析質譜儀的分析,發現內生菌菌絲體的類固醇成分相似於松露子實體。其中有兩種類固醇 (barrigenol R1、dehydroepiandrosterone)是第一次在松露中被發現。 第三部分研究以灰白松露(Tuber borchii)液態醱酵生產總三萜,研究結果,在不同碳氮源測試實驗中,以葡萄糖和malt extract為最佳碳氮源,當以葡萄糖濃度為20 g/L時,可得到較高的總三萜量71.17±3.05 mg/L,當以malt extract 濃度為20 g/L時,可得到較高的總三萜量123.27±4.22 mg/L。為了提升總三萜產量,另外添加不同油脂於培養基中,結果顯示以添加2 %的花生油有最佳的產量,其總三萜量為191.09±5.34 mg/L,為控制組(123.27±4.22 mg/L)之總三萜量的1.55倍。
The submerged fermentation process for the cultivation of mushroom mycelia and its secondary metabolites has been widely used for the production of pharmaceutical mushrooms. The mushroom’s metabolites could provide antitumor, hepatoprotective and pharmacological effects in the medical applications. In the first part of this study, exopolysaccharides (EPS) produced from Hypocreales sp. NCHU01 was carried out in the submerged cultivation. To find the optimal conditions, factors such as carbon and nitrogen source concentrations, pH, temperature, magnesium ion, and potassium ion levels were considered. The results showed that the optimal conditions were as follows: sucrose, yeast extract, magnesium sulfate and potassium dihydrogen phosphate were 10.6, 10.92, 1.0 and 1.01 g/L, respectively. The optimization conditions were set at 25 C with an initial pH 6.5. The maximum EPS production was about 1329 ± 23 mg/L, which was 3.34-fold to that of the original condition. The study displayed an increasing molecular weight distribution in the harvested EPS over a longer cultivation time. The quantitative relationship between mycelial biomass and EPS production was also built. In the second part, biomass and triterpenoids of the truffle endophytic fungus Hypocreales sp. NCHU01 were produced in the submerged culture. In the carbon source test, 10 g/L of sucrose yielded the highest triterpenoid production, reaching 132.26±1.77 mg/L. In the nitrogen source test, 15 g/L of yeast extract was identified as the most favorable source for crude triterpenoid production, reaching 157.68 mg/L. In the stimulatory test, the addition of 2 g/L linoleic acid or 125 mg/L chitosan to the medium was found to elevate triterpenoid production to 327.5 mg/L and 343.2 mg/L, respectively. The major sterol compositions of the fruiting body of Tuber magnatum and mycelia of Hypocreales sp. NCHU01 were identified by gas chromatography with a mass spectrometer. It was found that sterol composition in the fermentation mycelia of Hypocreales sp. NCHU01 was quite similar to that of the fruiting bodies of Tuber melanosporum, Tuber magnatum and Tuber sinense. Two kinds of sterols (i.e. barrigenol R1; and dehydroepiandrosterone) were identified for the first time from Tuber species. Finally, triterpenoids produced from Tuber borchii was carried out in the submerged cultivation. In the carbon source test, 20 g/L of glucose yielded the highest triterpenoid production (71.17±3.05 mg/L). In the nitrogen source test, 20 g/L of malt extract was identified as the most favorable source for crude triterpenoid production (123.27±4.22 mg/L). In the stimulatory effect, the addition of 2 % peanut oil to the medium was found to elevate triterpenoid production to 191.09±5.34 mg/L, which was 1.55-fold to that of the original condition.
其他識別: U0005-1308201516261700
文章公開時間: 2018-08-20
Appears in Collections:化學工程學系所



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