巴西洋菇液態發酵生產菌絲體及其多醣類

Production of Agaricus blazei Murrill and its Polysaccharide by Submerged Fermentation

林志輝(Jr-Hui Lin)

指導教授 ： 楊盛行

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00992

摘要

巴西洋菇為一巴西當地居民的野生食用菇，過去十年以來，巴西洋菇經由深入的研究與開發，已為東南亞健康食品市場上一種兼具有營養、嗜好和生理調節三種機能的新興食品。因各地所生產之巴西洋菇子實體在形態上已有差異，而且形態分類上也已經出現爭議，在進行有關研究之前，必須確認實驗材料物種。本研究由市售巴西洋菇子實體中成奶擢髐Q四株菌株，並以種專一性PCR 進行供試菌株及子實體的菌種鑑定，所有樣品之種專一性PCR 均得到預期的單一 DNA產物片段，其序列顯示相當高之相似性，並且與基因庫內之序列進行比對結果高度相符，確認供試材料為巴西洋菇。本研究以螢光核酸染劑 acridine orange對玻片培養三天之巴西洋菇菌絲進行染色，於螢光顯微鏡下可清楚觀察細胞與細胞核之形態，建立一螢光真菌菌絲形態及細胞核染色法。其中細胞質呈現橘紅色螢光而細胞核為綠色螢光，菌絲無扣子體，核數不規則，以四個最為常見，菌絲間似有細胞融合之現象。本研究以三種任意引子（R4、OPB15、OPB18）對供試菌株之 total DNA 進行放大，建立RAPD 圖譜。類緣樹狀圖將菌株分為主要三群，而巴西洋菇分離株M152 以及A321 與其他菌株類緣關係較遠，獨立不成群，其分別在多醣以及形態上與其他菌株顯著不同，顯示本研究依據RAPD 所建立之類緣關係反映了相當菌株特性。本研究以搖瓶培養方式探討巴西洋菇液態發酵培養之條件，巴西洋菇最適培養基組成為每公升培養基含有麥芽抽出物50克、酵母抽出物1克、peptone 5克。最適培養溫度為27oC，起始pH為6，培養體積及搖瓶轉速為在500毫升有溝三角瓶中加入200毫升培養基，先於90rpm 下培養3天後再以105rpm 培養5天。經由培養條件的最佳化，巴西洋菇分離株M21 的菌絲體產率由初始的6.63 ±0.41 克增加至10.33 ±0.08 克，相當於增加55.8％。巴西洋菇菌絲體乾重最高者為分離株M72 產率為每公升培養基 10.83 ±0.24克。多醣粗萃取物產率最高者為巴西洋菇分離株M152，產率為 3.03 ±0.05％，每公升培養基可獲得0.251 ±0.004克多醣粗萃取物。多醣粗萃取物之蛋白質含量低於1.63％，總醣含量82.27∼99.14％，HPGPC 分析顯示多醣粗萃取物之分子量組成約為274.1kDa、32.7kDa、7.5kDa 及2.1 kDa。

關鍵字

多醣 ； 液態發酵 ； 巴西洋菇 ； 螢光染色

並列摘要

Agaricus blazei Murrill is an edible mushroom originally used as folk remedy and food in Brazil. Over the last decade, A. blazei Murrill has been well studied and now developing as a novel functional food in Japan, Korea, China and Taiwan. The argument raised from morphological difference between A. blazei Murrill and A. blazei Heinem makes species-specific PCR is necessary before conduct subjects into test. In this study, 14 A. blazei Murrill strains was isolated from fruiting bodies, identified by species-specific PCR, established phylogenic tree by RAPD (random amplified polymorphic DNA) and optimized submerged fermentation by shaking flask fermentation. A new staining method that uses acridine orange to stain fungal mycelium and nucleus was established. A. blazei Murill mycelium has no clamp connection, with multi-nuclear(most are 4) cells. The phylogenic tree generated from RAPDs showed three groups and two distinct A. blazei Murrill strain M152 and A321 were isolated from all other strains. The optimized medium composition was 5% malt extract, 0.1% yeast extract and 0.5% peptone, pH 6. The optimized condition for submerged fermentation of A. blazei Murill was 27℃, 200mL optimal medium in 500mL hinton flask, incubated at 90 rpm for 3days then 105 rpm for 5days. The highest productivity of mycelium was 10.83±0.24g cell dry weight/L medium (A. blazei Murrill isolate M72). The highest yield of polysaccharide crude extract was 3.03±0.05 %, or 0.251±0.004 g/L medium(A. blazei Murrill isolate M152). All polysaccharide crude extracts’protein content was below 1.63% and total sugar content between 82.27∼99.14％. Polysaccharide crude extracts mainly consist of four different molecular weight components: 274.1kDa、32.7kDa、7.5kDa and 2.1 kDa. The 2.1 kDa portion should be a b-(1,3)-glucan.

並列關鍵字

polysaccharide ； submerged fermentation ； fluorescent microscopy ； Agaricus blazei

參考文獻

黃雅惠、雪蝎說C2004。利用核糖核酸基因序列進行巴西蘑菇親緣關係之探討。臺灣農業化學與食品科學，42(2):75-82

黃雅惠。1999。巴西洋菇分子生物鑑定系統之研究。碩士論文。國立臺灣大學農業化學所，台北，台灣。

Atkinson, B. and I. Daoud. 1976. Microbial flocs and flocculation in fermentation process engineering. Advances in Biochemical Engineering 4:41-124.

Barbisan, L. F., M. Miyamoto, C. Scolastici, D. M. F. Salcadori, L.R. Ribeiro, A. F. Eira and J. L. V. de Camargo. 2002. Influence of aqueous extract of Agaricus blazei Murill on rat liver toxicity induced by different doses of diethylnitrosamine. Journal of Ethnopharmacology 83:25-32.

Barreca, G. S., M. Giovanni, M. D. Massimo, L. Angelo, M. C. Liberto and A. Foca. 2000. Early detection of Leishmania promastigotes in dog bone marrow cultures by acridine orange stain. Diagnostic Microbiology and Infectious Disease 37(4):247-251.