培養條件對紫芝與赤芝菌絲生成多醣與靈芝酸含量與組成的影響

靈芝為中國傳統食用藥材，紫芝(Ganoderma formosanum)為臺灣特有種，而赤芝(Ganoderma lucidium)為常見之藥材及保健食品，被廣為研究及有效成分為靈芝酸以及多醣。本研究目的為建立一套靈芝酸分析平台，使用液相層析串聯質譜(HPLC-MS/MS)分析靈芝酸的種類，在特定能量下結因構不同使得斷片模式有所差異；另外使用高解析質譜(HRMS)得精確分子量以及紫外光吸收來推測靈芝酸種類以及含量。另一個具有生理活性成分為多醣體，其中廣泛被認為具有調節免疫結構之多醣體為(1,3;1,6)-β-D-葡聚醣分支度介於0.2-0.33，本實驗使用專一性酵素水解(1,3;1,6)-β-D-葡聚醣可精確得知含量並得知分支度。子實體生長時程曠日費時，但是液態醱酵具有培養時間快，分離萃取容易等優點。許多文獻研究指出靜置培養靈芝使得靈芝酸的產量增加，因此本實驗使用靜置培養方式產生靈芝酸及(1,3;1,6)-β-D-葡聚醣。搖瓶培養下菌絲體與多醣體生產較為快速以及大量，兩階段培養方式有利生產目標產物。本實驗研究目的在比較紫芝以及赤芝在三種不同培養方式下，菌絲體生成量、菌絲體中多醣體、醱酵液中多醣體、(1,3;1,6)-β-D-葡聚醣含量及其分支度以及靈芝酸含量與其種類的差異。結果顯示紫芝在靜置培養下菌絲體最多，並且第十天下菌絲體及醱酵液中多醣體最多，赤芝則是在搖瓶培養第十五天下菌絲體最多，並且菌絲體多醣體在第十天最多，但是醱酵液並沒有顯著差異。紫芝產(1,3;1,6)-β-D-葡聚醣，在靜置培養下第三十天含量最高並且顯著高於赤芝，並且分支度落於0.2-0.33之間，但是赤芝只有在靜置培養下才有(1,3;1,6)-β-D-葡聚醣產生並且分支度並低於0.2-0.33。整體而言赤芝菌絲體含量以及多醣含量高於紫芝，但是紫芝所產多醣體中(1,3;1,6)-β-D-葡聚醣佔多醣體比例高於赤芝。使用超高效能液相串聯質譜儀(UHPLC-MS/MS)靈芝酸分析方法平台中，從松杉靈芝以及赤芝子實體使用酒精萃取靈芝酸液中建立品管樣品，品管樣品中推測可能含有靈芝酸27種，並且使用標準添加法測品管樣品中，具有靈芝酸標準品種類的真實濃度以及相對定量無標準品之靈芝酸，另外回收率推測質譜訊號影響樣品基質的結果收吸波峰的特徵篩選波峰，進一步利用高解析質譜(HRMS)分析其精確分子量，此方法理論分子質荷比(m/z)與實際測試m/z的偏差值在-2.33~ 2.30 ppm之間。最後在本實驗培養方法下，兩株菌在三種培養方式下以紫芝靜置培養30天生成Ganoderi acid H 0.99 ppm最多，並且高於赤芝搖瓶培養的0.23 ppm，赤芝靜置培養以及搖瓶培養下生成Ganoderic acid H 則無顯著差異。

關鍵字

Ganoderma formosanum ； Ganoderma lucidium ； LCMS/MS ；靈芝酸； (1,3 ； 1,6)-β-D-葡聚醣；分支度；多醣

並列摘要

Ganoderma formosanum and Ganoderma lucidium, Lingzhi, are traditional Chinese medicine and are considered as healthy food. The bioactive components in Lingzhi are (1, 3；1, 6)-β-D-glucans and ganoderic acids. This study aims to investigate that the effect of the static culture, the submerged culture and the two-stage fermentation on the production of the (1, 3；1, 6)-β-D-glucans and ganoderic acids of Ganoderma formosanum and Ganoderma lucidum. The biomass production, polysaccharide in mycelium and broth, the content and the degree of the branch of (1,3；1,6)-β-D-glucans and the structure of ganoderic acids of Ganoderma formosanum and Ganoderma lucidum were compared. In this study, the (1,3)-β-D-glucanase was used to investigate the content and the degree of the branch of (1,3；1,6)-β-D-glucans. Liquid chromatography-tandem mass spectrometry was used to analyze ganoderic acids. The structure properties of ganoderic acids were identified by accurate m/z (-2.33-2.33 ppm), MS/MS spectra and UV spectra. Standard addition method was used to analyze the concentration of ganoderic acids. Relative quantification method was used to analyze the ganoderic acids without an available commercial standard. In this research, the result shows that static culture produces more ganoderic acid than submerge culture. However, shaking culture produces more biomass and polysaccharide. Two-stage fermentation can enhance the yield of the target compound. The mycelium of Ganoderma formosanum has maximum biomass by static culture. At 10 days, Ganoderma formosanum has the maximum yield of polysaccharides in the mycelium and the broth. By shaking culture, the maximum biomass of the mycelium of Ganoderma lucidum is at 15 days and the maximum yield of the polysaccharide of Ganoderma lucidum is at 10 days but there is no significant difference in the broth. At 30 days, by static culture, the content of (1,3；1,6)-β-D-glucan of Ganoderma formosanum mycelia is more than Ganoderma lucidum. The degree of the branch of (1,3；1,6)-β-D-glucan of Ganoderma formosanum mycelia is between 0.2-0.33. Ganoderma lucidum only produces (1,3；1,6)-β-D-glucan in static culture and the degree of the branch of (1,3；1,6)-β-D-glucan is lower than 0.2-0.33. Ganoderma lucidum contains more mycelia and polysaccharides than Ganoderma formosanum. However, the percentage of (1,3；1,6)-β-D-glucan in the polysaccharides of Ganoderma formosanum is more than Ganoderma lucidum. Ganoderma lucidum and Ganoderma tsugae fruiting body were used as QC sample. 27 kinds of ganoderic acids were found in QC sample. In this study, the mycelium of Ganoderma formosanum in static culture 30 days has more ganoderic acid H than Ganoderma lucidum. The static culture and shaking culture have no significant effect on the production of Ganoderic acid H of Ganoderma lucidum.