臺灣博碩士論文加值系統

本研究探討以耐糖度最高且耐高滲透壓之酵母菌( Saccharomyces
rouxii ) 生產葡萄糖耐糖因子，生產菌株篩選用來自醬醪所篩選出，菌株篩選以比生長速率、鉻離子消耗速率為篩選依據指標，所得菌株做為各種操作變因之探討。本研究比較靜置發酵與噴流供氧( Sparging )方式以及間歇加壓供氧方式，溶氧動力學顯示在2.5-10 psi 壓力情況下，以噴流供氧( Sparging )方式測得水中飽和溶氧量5 秒鐘即可達到飽和 (6 ppm)。間歇加壓供氧方式所需較長時間為20 分鐘即達飽和。間歇加壓供氧方式可避免發酵過程中，噴流供氧所產生之起泡現象與添加酵泡劑之困擾。考慮酵母菌生長速率倍增時間 ( doubling time )約為1~2 小時，間歇加壓供氧方式培養為可行之選項。本研究所得之結論，利用間歇加壓供氧方式模擬發酵培養，
最佳培養條件：發酵液500 mL、接菌體積1 %、培養溫度30 ℃、起始酸鹼值4.0、Cr3+濃度100 ppm、葡萄糖15 % 通氣壓力2.5 psi 、通氣時間 20 分鐘、洩壓 3 秒鐘；32 h 後，Cr3+與葡萄糖濃度均降至零，酵母菌細胞為2.5×108 CFU/mL。

This research investigated the production of glucose tolerance factor (GTF)by a highly osmotic and sugar tolerant yeast (Saccharomyces rouxii). The strain
was screened and isolated from soy sauce mash. Strain selection was based on relative specific growth rate and chromium consumption rate. The obtained strain was used for studying various operation variables. The dissolved oxygen
(DO) in static culture, air sparging and intermittent pressurized air of operation modes was compared. Results showed that, at room temperature, DO could reach saturation (6 ppm) within 5 sec, while intermittent pressurized air took 20 min to reach saturation. Using intermittent pressurized air system, bubbling and the need of antifoaming agent, as caused by sparging air, can be
avoided.Considering the doubling time of yeast (1~2 h), the intermittent pressurized air system is a practical option. Experimental results showed that using intermittent pressurized air system, for a working volume of 500 mL,
initial 100 ppm of Cr3+ and 15% glucose were completely consumed after 32 h. The culture temperature was 30 °C, initial pH was 4.0, and the air pressure was 2.5 psi. The fermenter was pressurized for 20 min and then released for 3 sec. The final yeast concentration was 2.5×108 CFU/mL.

中 文 摘 要 ...................................... I
Abstract ......................................... I
誌謝 ............................................. II
目錄 ............................................. III
圖目錄 ........................................... VIII
表目錄 ........................................... XII
第一章 緒論 ...................................... 1
第二章 文獻回顧 .................................. 2
2.1 前言 ......................................... 2
2.2 糖尿病的種類 ................................................... 2
2.2.1 第一型糖尿病 ................................ 2
2.2.3 其他型糖尿病 ................................ 3
2.2.4 妊娠型糖尿病 ................................ 4
2.3 糖尿病治療 .................................... 4
2.3.1 胰島素 ...................................... 4
2.3.2 口服藥物 .................................... 5
2.3.3 金屬離子 .................................... 6
2.4 鉻的化學性質 .................................. 8
2.5 鉻的循環與利用 ................................ 8
2.5.1 鉻的吸收 .................................... 9
2.5.2 鉻在器官的分部............................... 10
2.5.3 鉻的代謝 .................................... 10
2.5.4 人體鉻的含量 ................................ 10
2.5.5 人類對鉻的需求............................... 11
2.7 葡萄糖耐糖因子 ................................ 13
2.7.1 GTF 之安全性 ................................ 16
2.8 菌種介紹 ...................................... 17
2.8.1 酵母菌之型態 ................................ 17
2.8.2 酵母菌之繁殖方法 ............................ 19
2.9 糖解作用 ...................................... 26
2.9.1 EMP 路徑 .................................... 26
2.10 固定化 ....................................... 30
2.10.1 固定化的定義及方法 ......................... 30
2.10.2 固定化方法之類型 ........................... 30
2.10.5 微生物固定化的技術與優點 ................... 36
2.11 氧氣傳遞 ..................................... 41
2.11.1 氧氣傳送之途徑與機制 ....................... 43
2.11.2.1 動態排氣量測法 ( Dynamic Gasing-out Method)... 45
第三章 材料與方法 ................................. 47
3.1 實驗材料 ...................................... 47
3.1.1 培養基 ...................................... 47
3.1.1.2 固態培養基 ................................ 47
3.1.1.2 基礎液態培養基 ............................ 47
3.1.2 化學藥劑 .................................... 47
3.2 實驗儀器 ...................................... 50
3.3.1 原子吸收光譜儀 ( Atomic absorption spectrometry,AAS ) ................................................... 52
3.3.1.1 火焰式原子吸收光譜儀基本構造............... 52
3.3.2.1 RI 偵測器 ................................. 54
3.4.1 實驗大鋼 .................................... 56
3.5. 1 菌種篩選 ................................... 57
3.5.2 菌相觀察 .................................... 61
第四章結果與討論 .................................. 77
4.1 酵母菌之基礎搖瓶培養 .......................... 77
4.2 酵母菌之生長曲線 ( growth curve ) ............. 77
4.3 酵母菌細胞大小之量測 .......................... 81
4.4 菌種篩選 ...................................... 83
4.5 酵母菌於水膠膜中之起始週其特性................. 85
4.6 酵母菌於不同溫度下之生長情形 .................. 88
4.7 酵母菌於不同震盪轉速下之探討 .................. 94
4.8 酵母菌於不同起始pH 值下之生長情形 ............. 100
4.9. 不同鉻濃度對酵母菌之吸附能力 ................. 104
4.10 間歇供氧方式培養酵母菌 ( Saccharomyces rouxii ) 發酵之探討............................................... 110
4.10.1 利用加壓供氧方式對水中DO 值的影響 .......... 110
4.10.2 通氣時間對水中DO 值之影響....................110
4.10.3 通氣壓力對水中DO 值之影響 .................. 113
4.10.4 加壓供氧發酵程序架構之建立 ................. 115
4.10.5 利用間歇加壓供氧方式模擬發酵培養 ........... 118
第五章 結論 ....................................... 125
第六章 參考文獻 ................................... 128
圖目錄
圖2-1 GTF 以三價鉻為中心結構 ............................................................. 14
圖2-2 酵母菌之基本型態 ........................................................................ 20
圖2-3 不同種屬酵母之型態 .................................................................... 24
圖2-4 Saccharomyces rouxii 型態圖 ..................................................... 25
圖2-5 EMP 路徑 ...................................................................................... 27
圖2-6 檸檬酸循環 ................................................................................... 29
圖2-7 吸附法 ( adsorption ) ..................................................................... 31
圖2-8 共價鍵結合法 ( covalent binding ) ................................................ 32
圖2-9 離子鍵結法 ( ionic bond ) ............................................................. 32
圖2-10 交聯法 ( cross-linking )................................................................ 33
圖2-11 包埋法 ( entrapment ) .................................................................. 33
圖 2-12 氣液質傳示意圖 .......................................................................... 44
圖2-13 溶氧電極 ( DO sensor ) 示意 ...................................................... 46
圖3-1 火焰式原子吸收光譜儀之基本構 ................................................. 52
圖3-2 能階示意圖 .................................................................................... 53
圖3-3 篩選具有GTF 活性之酵母流程圖 ................................................ 60
圖3-4 血球計數器示意圖 ......................................................................... 61
圖3-5 血球計數器 .................................................................................... 66
圖3-6 以HPLC 測定甲醇標準品，滯留時間 14.26 分鐘， ................ 72
圖3-7 玻璃表面之反應情形 ..................................................................... 73
圖3-8 投影片於玻璃夾層中 ..................................................................... 76
圖3-9 注入水膠於玻璃夾層中 ................................................................. 76
圖3-10 水膠膜於紫外燈光下照射 ........................................................... 76
圖4-1 酵母菌Saccharomyces rouxii 不同時間之生長情形 ..................... 78
圖4-3 分光光度計OD600 操作示 ........................................................... 79
圖4-2 改良式三角錐形瓶 ......................................................................... 79
圖4-4 酵母菌生長曲線( growth curve )圖 ................................................ 80
圖4-5 Saccharomyces rouxii 於顯微鏡下 ( 400 倍 ) 之示意圖。 ....... 81
圖4-6 實驗型拍攝鏡頭與像位差顯微鏡架設情形 .................................. 82
圖4-7 固定化Saccharomyces rouxii 第一次發酵與第二次發酵以及free
cell 情況下之發酵液濁度變化 ...................................................... 86
圖4-8 顯微鏡 ( 400 倍 ) 酵母菌於水膠膜內部之生長情形 .................. 87
圖4-9 不同溫度條件下Saccharomyces rouxii 之菌體乾重變化 ( Free
cell )。 ........................................................................................... 90
圖4-10 不同溫度條件Saccharomyces rouxii 之GTF 活性變化 .............. 91
圖4-11 不同溫度條件下Saccharomyces rouxii 之菌體乾重變化 (菌體固
定化第二次發酵 )。 ..................................................................... 92
圖4-12 不同溫度條件下 Saccharomyces rouxii 之GTF 活性變化( 菌體固
定化第二次發酵 ) ......................................................................... 93
圖4-13 不同轉速下Saccharomyces rouxii 之菌體乾重變化 (Free cell) 96
圖4-14 不同震盪轉速條件下 Saccharomyces rouxii 之GTF 活性變化
( Free cell ) ..................................................................................... 97
圖4-15 不同轉速下Saccharomyces rouxii 之菌體乾重變化 (菌體固定化
第二次發酵) .................................................................................. 98
圖4-16 不同震盪轉速條件下 Saccharomyces rouxii 之GTF活性變( 菌體
固定化第二次發酵 ) ..................................................................... 99
圖4-17 不同起使pH 值Saccharomyse rouxii 之生長情形 ................... 102
圖4-18 不同起使pH Saccharomyse rouxii 之GTF 活性變化 ............ 103
圖4-19 不同鉻濃度固定化第一次發酵Saccharomyces rouxii 之變化菌體
乾重 ............................................................................................. 106
圖4-20 不同鉻濃度固定化第二次發酵Saccharomyces rouxii 之變化 (A)
菌體乾重 (B) GTF 活性。 ......................................................... 107
圖4-21 不同鉻濃度固定化第三次發酵Saccharomyces rouxii 之變化 (A)
菌體乾重 (B) GTF 活性。 ......................................................... 108
圖 4-22 不同鉻濃度固定化第四次發酵Saccharomyces rouxii 之變化 (A)
菌體乾重 (B) GTF 活性。 ......................................................... 109
圖4-23 動態排氣量測法 ( Dynamic Gasing-out Method ) 裝置圖 ........111
圖4-24 以Sparging 通氣方式對水中DO 值之影響 ............................. 112
圖4-25 通氣時間對水中DO 值之影響 .................................................. 114
圖4-26 小型模擬發酵槽的程序架構 ..................................................... 116
圖4-27 間歇加壓供氧發酵槽裝置圖 ..................................................... 117
圖4-28 間歇性加壓供氧發酵槽氣體流動方向與設備 .......................... 120
圖4-29 Saccharomyse rouxii 於間歇性供氧發酵槽之生長情形 ( A ) 0
hr ( B ) 16 hr ( C ) 32 hr ( D ) 40 hr .............................................. 121
圖4-30 以間歇性加壓供氧方式培養Saccharomyse rouxii 之細胞生長曲
線圖 ............................................................................................. 122
圖4-31 以間歇性加壓供氧方式培養Saccharomyse rouxii 之細胞生長曲
線與三價鉻濃度的消耗 .............................................................. 123
圖4-32 以間歇性加壓供氧方式培養Saccharomyse rouxii 之葡萄糖消耗
與乙醇之生成關係圖 .................................................................. 124

表目錄
表2-1 各類飲食中所含之鉻含量 ............................................................. 12
表2-2 各種人工合成固定化法之比較 ..................................................... 34
表2-3 固定化材料種類分類表 ................................................................. 35
表2-4 各式固定化方法之優缺點比較 ..................................................... 40
表 2-5 空氣中的氧在水中的溶解度 ........................................................ 42
表3-1 YMP 固態培養基成分 ................................................................... 49
表3-2 YMP 液態培養基成分 ................................................................... 49

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