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研究生:李微萱
研究生(外文):Li wei-shuan
論文名稱:液態培養條件對舞菇(Grifolafrondosa)菌絲體及多醣體生產的影響
論文名稱(外文):Effect of submerged culture conditions on the production of mycelial biomass and polysaccharides of Grifola frondosa.
指導教授:顏文義顏文義引用關係
指導教授(外文):Wen-Yi Yen
學位類別:碩士
校院名稱:東海大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:99
中文關鍵詞:舞菇多醣體β﹣1→3﹣D﹣glucan氣舉式發酵槽攪拌式發酵槽
外文關鍵詞:Grifola frondosapolysaccharideβ-1→3-glucansstirred tank fermemtorair-lift fermentor
相關次數:
  • 被引用被引用:9
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  • 下載下載:1281
  • 收藏至我的研究室書目清單書目收藏:2
舞菇(Grifola frondosa)為一種食藥用的真菌,代謝產物—多醣體,有抗腫瘤和保肝的療效,而以具有β﹣1→3﹣及β﹣1→6﹣為鍵結的葡聚醣,最具有藥理功效,故對於如何以人工培養舞菇並獲得多醣體值得加以探討。本研究以搖瓶及發酵槽培養的方式來探討舞菇菌絲生長及其多醣體的產量。
舞菇在搖瓶實驗發現glucose、fructose、sucrose及corn starch四種碳源中,以30g/L的corn starch能得到最好的菌絲生長及多醣體產量。氮源以6g/L的corn steep powder (CSP) ,就菌體及胞外多醣體的產量而言,優於yeast extract與malt extract,但胞內多醣體含量則是CSP及malt extract是一樣的。培養基初始pH值以pH 6有較佳的多醣產量,可得到菌絲乾重1.93mg/mL、胞內多醣1.28mg/mL以及胞外多醣10.69mg/mL的產量,pH5的效果次之。
以7L的攪拌式發酵槽經過十四天的培養,發現三種通氣量中(0.5,1.0,1.5vvm),以1.0 vvm可以得到最佳的菌絲重量1.43 mg/mL,胞內多醣1.49mg/mL,以及胞外多醣14.25mg/mL的產量﹔以1.5 vvm所得的多醣體含有最多胞外β﹣1→3﹣D﹣glucans含量111.90μg/mL LE﹔以1.0 vvm則可以得到胞內多醣體中最多的β﹣1→3﹣glucans的含量10.45μg/mL LE。
以7L的氣舉式發酵槽經過十四天的培養,以0.5 vvm可以得到最佳的菌絲重量1.33 mg/mL,以及胞外多醣12.69mg/mL的產量﹔IPS以1.0 vvm的1.41 mg/mL較佳,所以氣舉式發酵槽中以0.5vvm是比較好的選擇,三種通氣量中,1.5 vvm所測得的多醣體含有最多胞外β﹣1→3﹣glucans含量40.14μg/mL LE,以及胞內多醣體中最多的β﹣1→3﹣glucans的含量11.56μg/mL LE。
經由凝膠透過層析法(Gel permeation chromatography)方法分析其分子量,舞菇多醣體經由七天發酵後,在攪拌式發酵槽中EPS是以0.5 vvm最大2.0x106 Da,IPS是以0.5 vvm所得到的分子量最大5.0x105 Da,1.5 vvm的分子量差距最多,5.3x103 Da到4.8x105 Da; 氣舉式發酵槽多醣體分子量經過七天的發酵,EPS以0.5 vvm最大3.5x105 Da,但是分佈差距也最大﹔IPS的分子量以1.0 vvm 所產生的分子量3.5x105 Da為最大。
Grifola frondosa is am edible medicinal fungi with medicinal effect in anti-tumor and liver protecting. The polysaccharide produced by the fungi having the chemical structure of β-1→3- andβ-1→6-glucans,is believed to have biological and pharmaceutical activities. Therefore,research in the cultivating Grifola frondosa for biomass and its polysaccharide production is in high demand.
Results show that shake flask fermentation the most suitable carbon source was corn starch at concentration of 30 grams per liter of medium for the polysaccharical for mation,as compared to glucose, fructose and sucrose, where the biomass, exo-polysaccharide (EPS) and intra-polysavvharide (IPS) were 1.70mg/mL, 7.74 mg/mL and 1.12 mg/mL ,and the use of corn steep powder is better than yeast extract and malt extract as N-source. At pH 6 is the best among 4 tested initial pH in fermentation media to have mycelium dry weight(MDW) 1.93 mg/mL EPS, 10.69 mg/mL, and 1.28 mg/mL IPS.
The 7 liter stirred tank fermentor(STF) studies indicate,aeration rate at 1.0vvm favors the maximum production in 1.43 mg/mL of MDW,14.25 mg/mL EPS,and 1.49 mg/mL IPS after eight days in the 7 liter fermentor while 1.5vvm obtained the highest β-1→3-glucans , 111.90μg/mL LE in the EPS.
The 7 liter the air-lift fermentor(ALF) studies, aeration at 0.5 vvm favors the production of the mycelium(1.33 mg/mL) and EPS (12.69 mg/mL ),while IPS was best with 1.0vvm(1.41mg/mL). Theβ-1→3-glucans content, in the EPS were similar in all there aeration rate in ALF(40.14μg/mL LE) ,but were higher in IPS(11.56 μg/mL LE),with the 1.5 vvm aeration the best.
The molecule weight (MW) of the polysaccharide after days 7 of fermentation, as analysed by gel permention chromatography (GPC) was the highest in the STF with 0.5 vvm (EPS: 2.0x106 Da;IPS: 5.0x105Da). After days 7 of fermentation on the air-lift fermentor was the EPS highest MW 3.5x105Da with 0.5vvm, and IPS highest MW 3.5x105Da with 1.0vvm.
中文摘要........................i
英文摘要.......................iii
第一章 緒論......................1
一、前言......................1
二、研究目的....................2
第二章 文獻回顧....................3
  一、舞菇簡介....................3
  二、舞菇的人工培養.................10
  三、發酵槽的介紹..................14
四、Aniline blue與β﹣1→3﹣D glucan的關係介紹..17
第三章 材料與方法...................18
  一、菌種及培養基..................18
  二、實驗流程....................19
  三、發酵槽構造...................19
  四、菌種保存及菌體培養...............21
  五、菌酛培養....................22
  六、搖瓶培養....................22
  七、發酵槽培養...................23
  八、分析方法....................24
九、統計分析....................28
第四章 結果與討論...................29
  一、搖瓶實驗探討..................29
(一) 不同的碳源培養對舞菇生長的影響.......29
(二) 不同的氮源培養對舞菇生長的影響.......31
(三) 不同corn starch濃度對舞菇生長的影響....34
(四) 培養基初始pH值對舞菇生長的影響.......35
  二、發酵槽培養實驗.................37
(一) 攪拌式發酵槽對舞菇生長的影響........38
(二) 氣舉式發酵槽對舞菇生長的影響........40
(三) 不同發酵槽環境下所生產的多醣體分子量比較討論43
第五章 結論......................47
第六章 參考文獻....................95
附錄
1. 多醣體分子量標準曲線................102
2. β-1→3﹣glucans含量標準曲線............102






表目錄

表一、舞菇金屬蛋白酶性質................50
表二、由舞菇菌絲體萃取得的抗腫瘤活性多醣........51
表三、由舞菇子實體萃取所得的抗腫瘤活性多醣.......52
表四、不同的碳源對於舞菇菌體生長及多醣體含量之影響...53
表五、不同的氮源對於舞菇菌體生長及多醣體含量之影響...54
表六、不同的初始碳源濃度對於舞菇菌體生長及多醣體含量之影55
表七、不同的初始pH值對於舞菇菌體生長及多醣體含量之影響.56
表八、不同通氣量對攪拌式發酵槽中舞菇之菌體濃度、多醣產量、及多醣所含β-1→3–glucans量的影響.........57
表九、不同通氣量在氣舉式發酵槽中培養舞菇對菌體濃度、多醣體產量及多醣中β-1→3–glucans含量的影響.......58
表十、在攪拌式發酵槽中不同通氣量對舞菇之多醣體分子量之影響........................59
表十一、氣舉式發酵槽中不同通氣量對舞菇之多醣體分子量分部的影響........................60


圖目錄

圖一(a)、舞菇子實體外觀.................61
圖一(b)、舞菇菌絲....................61
圖二、β-1→3、β-1→6-D-葡聚醣立體結構.........62
圖三、有抗腫瘤活性的β-1→3-D-葡聚醣結構.........63
圖四、真菌細胞壁模型...................64
圖五、β-D 葡聚醣多醣體的抗腫瘤作用之可能機制......65
圖六(a)、標準式渦輪扇葉﹔(b)、船推進器型扇葉.......66
圖七、氣舉式發酵槽槽內之導流管..............67
圖八、經由劃線培養活化的舞菇菌絲 (第七天)........68
圖九、經由再次活化的舞菇菌絲 (第七天)..........68
圖十、在三角瓶內的舞菇菌酛(剛接種)............69
圖十一、舞菇經過七天搖瓶培養之菌絲型態 (第七天).....69
圖十二、不同碳源對舞菇的(a)菌絲體生長及(b)發酵液還原糖和殘留澱粉的影響...................70
圖十三(a)、舞菇以glucose和corn starch為碳源 進行搖瓶實驗的形態--- glucose (第一天)............71
圖十三(b)、舞菇以glucose和corn starch 為碳源進行搖瓶實驗的形態--- corn starch (第一天)..........71
圖十四、不同碳源對舞菇發酵期間胞內多醣及胞外多醣產量的影響.......................72
圖十五、不同碳源對舞菇發酵期間的pH變化.........73
圖十六、不同氮源對舞菇發酵期間(a)菌絲體生長(b)發酵液還原糖的影響......................74
圖十七、不同氮源對舞菇發酵期間澱粉殘留量的影響......75
圖十八、不同的氮源對舞菇多醣含量的影響..........76
圖十九、不同氮源對舞菇發酵期間pH值和黏度的影響.....77
圖二十、不同玉米澱粉濃度對舞菇發酵期間(a)菌絲體生長及(b)發酵液還原糖的影響.................78
圖二十一、不同玉米澱粉濃度對舞菇發酵期間澱粉殘留量的影響.79
圖二十二、不同玉米澱粉濃度對舞菇發酵期間多醣含量之影響..80
圖二十三、不同初始pH值對舞菇發酵期間(a)菌絲體生長及(b)發酵液還原糖變化的影響...............81
圖二十四、不同初始pH值對舞菇發酵期間澱粉殘留量的影響..82
圖二十五、不同初始pH值對舞菇發酵期間多醣含量之影響...83
圖二十六、不同初始pH值對舞菇發酵期間(a)pH值和(b)黏度的變影響.......................84
圖二十七、不同通氣量在攪拌式發酵槽培養舞菇對其菌體乾重發酵
液還原糖的影響................85
圖二十八、不同通氣量在攪拌式發酵槽培養舞菇對其多醣體含量的
影響.....................86
圖二十九、舞菇在攪拌式發酵槽中培養不同通氣量對其β﹣1→3﹣D
﹣glucans含量的影響............ .87
圖三十、不同通氣量在攪拌式發酵槽培養舞菇對其pH值和黏度的變化........................88
圖三十一、舞菇在攪拌式發酵槽培養期間其胞外多醣、菌絲重、還原糖變化量、殘澱粉量、黏度及pH值的變化.....89
圖三十二、不同通氣量在氣舉式發酵槽對舞菇菌體生長及發酵液還原糖的影響....................90
圖三十三、氣舉式發酵槽中不同通氣量對培養舞菇多醣體產生的
影響......................91
圖三十四、在氣舉式發酵槽中以不同通氣量培養舞菇對其β﹣1→3﹣D﹣glucans含量的影響............92
圖三十五、不同通氣量在攪拌式發酵槽培養舞菇對其pH值和黏度的變化......................93
圖三十六、舞菇在氣舉式發酵槽培養期間其胞外多醣、菌絲重、還原糖變化量、殘澱粉量、黏度及pH值的變化影響....94
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