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研究生:張玉芝
研究生(外文):Yu-Chih Chang
論文名稱:利用固定化RhodococcusequiNo.23細胞生產膽固醇氧化酶
論文名稱(外文):Production of cholesterol oxidase with immobilized cells of Rhodococcus equi No. 23
指導教授:周正俊周正俊引用關係
指導教授(外文):Cheng-Chun Chou
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
畢業學年度:88
語文別:中文
論文頁數:74
中文關鍵詞:固定化膽固醇氧化酶海藻酸鈣
外文關鍵詞:immobilizationcholesteol oxidasealginateRhodococcus equi No.23
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中文摘要
Rhodococcus equi No. 23能產生膽固醇氧化酶(Cholesterol Oxidase, CO),可應用於食品與血液中膽固醇含量之測定,亦可分解膳食中之膽固醇以製造低膽固醇含量之食品。本研究乃在探討利用海藻酸鈣固定化R. equi No. 23以生產CO。結果顯示,固定化過程中對菌體並不會造成傷害。接種菌量、培養液起始pH值、培養溫度及振盪速率均會影響固定化R. equi No. 23之生長及CO之產生。固定化菌體產生CO之最適培養液起始pH值、培養溫度及震盪速率分別為7.0、30℃及250 rpm。在最適條件下,接種14.0 g/100 mL之固定化膠粒於培養液中,經48小時之培養後,CO活性達0.93 unit/mL。以固定化R. equi No. 23進行每批次48小時之重複批式發酵,經四批次後,CO產生之活性仍達0.76 unit/mL,約為第一批次產量之87 ﹪。

Abstract
Cholesterol oxidase (CO) was produced by Rhodococcus equi No. 23, it has been used for the determination of cholesterol in food and blood serum. In addition, CO can also be employed to degrade dietary cholesterol to produce low-cholesterol food. This study was conducted to investigate the production of CO by immobilized cells of R. equi No. 23. No detrimental effect was found on R. equi No. 23 during the immobilization procedures using sodium alginate. Inoculum size, initial pH of medium, cultivation temperature, and shaking speed affected the growth and CO production by immobilized R. equi No. 23. The optimal initial pH of culture medium, incubation temperature and shaking speed for the production of CO by the immobilized cell were found to be at pH 7.0, 30℃and 250 rpm, respectively. After 48 h of cultivation under the optimal conditions, a CO activity of 0.93 unit/mL was noted in the medium, which was inoculated with 14.0 g/100 mL cell-containing gel beads. Reuse of cell-containing gel beads for repeated batch fermentation at 48-hour intervals revealed that CO production in the 4th cycle was 0.76 unit/mL, about 87 ﹪of that in the first cycle.

目 錄
頁次
中文摘要---------------------------------------------------------------------------------- Ⅰ
英文摘要---------------------------------------------------------------------------------- Ⅱ
一、前言---------------------------------------------------------------------------------- 1
二、文獻整理---------------------------------------------------------------------------- 2
2.1膽固醇氧化酶之介紹------------------------------------------------------------- 2
2.1.1膽固醇的一些理化性質---------------------------------------------------- 2
2.1.2膽固醇氧化酶之反應機制------------------------------------------------- 3
2.1.3膽固醇氧化酶之應用------------------------------------------------------- 4
2.2膽固醇氧化酶之生產菌株------------------------------------------------------- 5
2.2.1 Rhodococcus equi No. 23菌株及其膽固醇氧化酶特性--------------- 6
2.2.2影響微生物生產膽固醇氧化酶之因子---------------------------------- 8
2.3固定化細胞------------------------------------------------------------------------- 9
2.3.1固定化之定義及應用------------------------------------------------------- 9
2.3.2固定化細胞的特點---------------------------------------------------------- 9
2.3.3固定化方法之原理---------------------------------------------------------- 12
2.3.4細胞固定化之材質---------------------------------------------------------- 14
2.3.5影響海藻酸鈣膠體強度之因子------------------------------------------- 16
2.3.6海藻酸鈣固定化細胞操作上的問題及解決之道---------------------- 19
三、材料方法---------------------------------------------------------------------------- 22
3.1 實驗材料--------------------------------------------------------------------------- 22
3.1.1菌種---------------------------------------------------------------------------- 22
3.1.2培養基------------------------------------------------------------------------- 22
3.1.3藥品---------------------------------------------------------------------------- 22
3.2 實驗方法--------------------------------------------------------------------------- 23
3.2.1菌種保存與活化------------------------------------------------------------- 23
3.2.2培養液製備------------------------------------------------------------------- 23
3.2.3 R. equi No. 23種菌生長曲線之測定------------------------------------- 23
3.2.4 R. equi No. 23菌體之固定化過程---------------------------------------- 23
3.2.5操作條件對固定化及游離菌體生長及cholesterol oxidase產量之 影響----------------------------------------------------------------------------
24
(1)種菌培養時間------------------------------------------------------------ 24
(2)以海藻酸鈉為包埋材料,各操作步驟對R. equi No. 23存活之影響---------------------------------------------------------------------
24
(3)接菌量--------------------------------------------------------------------- 24
(4)培養液起始pH值------------------------------------------------------- 25
(5)不同培養溫度------------------------------------------------------------ 25
(6)靜置及不同振盪速率--------------------------------------------------- 25
3.2.6電子顯微鏡觀察固定化細胞膠粒---------------------------------------- 25
3.2.7 R. equi No. 23固定化及游離細胞生產曲線測定---------------------- 26
3.2.8固定化細胞膠粒之重複使用性------------------------------------------- 26
3.2.9分析方法---------------------------------------------------------------------- 26
(1)菌數測定------------------------------------------------------------------ 26
(2) pH值測定---------------------------------------------------------------- 27
(3)膽固醇氧化酵素活性之測定--------------------------------------- 27
3.2.10統計分析--------------------------------------------------------------------- 28
四、結果與討論------------------------------------------------------------------------- 29
4.1 R. equi No. 23之生長曲線------------------------------------------------------- 29
4.2 操作條件對固定化及游離R. equi No. 23菌體生長與cholesterol oxidase產量之影響--------------------------------------------------------------
29
(1)不同預培養時間菌體及固定化後菌體之生長及膽固醇氧化之生產---------------------------------------------------------------------
29
(2)以海藻酸鈉為包埋材料,各操作步驟對R. equi No. 23存活之影響---------------------------------------------------------------------
34
(3)以掃瞄式電子顯微鏡觀察海藻酸鈣膠粒之結構與膠粒內R. equi No. 23分佈情形---------------------------------------------------
38
(4)不同接菌量對固定化及游離R. equi No. 23生長及CO生產之影響------------------------------------------------------------------------
38
(5)培養液不同起始pH值對固定化及游離R. equi No. 23生長及CO生產之影響----------------------------------------------------------
43
(6)不同培養溫度對固定化及游離R. equi No. 23生長及CO生產之影響---------------------------------------------------------------------
48
(7)不同振盪速率對游離及固定化R. equi No. 23生長及CO生產之影響---------------------------------------------------------------------
52
4.3游離及固定化R. equi No. 23培養過程中菌體之生長及CO活性之變化------------------------------------------------------------------------------------
56
4.4海藻酸鈣固定化R. equi No. 23膠粒之重複使用--------------------------- 59
五、結論---------------------------------------------------------------------------------- 61
六、參考文獻---------------------------------------------------------------------------- 63
圖 次
頁次
圖一 固定化方法---------------------------------------------------------------------- 13
圖二 海藻酸鈣之固定化裝置------------------------------------------------------- 17
圖三 游離R. equi No. 23於培養過程中菌體之生長與培養液pH值之變化----------------------------------------------------------------------------------
30
圖四 取不同培養時間之R. equi No. 23培養液進行固定化,經48小時培養後菌體生長情形----------------------------------------------------------
32
圖五 取不同培養時間之R. equi No. 23培養液接種至培養基中,經48小時培養後菌體生長情形----------------------------------------------------
33
圖六 取不同培養時間之R. equi No. 23培養液進行固定化,經48小時培養後,對於膽固醇氧化生產之影響----------------------------------
35
圖七 以海藻酸鈣作為固定化R. equi No. 23之材料,操作過程菌體之存活情形----------------------------------------------------------------------------
37
圖八 掃瞄式電子顯微鏡圖片------------------------------------------------------- 39
圖九 不同接菌量對游離R. equi No. 23於培養液中生長及膽固醇氧化生產之影響----------------------------------------------------------------------
41
圖十 不同接菌量對固定化R. equi No. 23於培養液中生長及膽固醇氧化生產之影響-------------------------------------------------------------------
42
圖十一 游離R. equi No. 23於不同培養液起始pH值下,培養48小時後菌體生長及膽固醇氧化生產之影響----------------------------------
45
圖十二 固定化R. equi No. 23於不同培養液起始pH值下,培養48小時後菌體生長及膽固醇氧化生產之影響-------------------------------
46
圖十三 游離R. equi No. 23於不同培養溫度下,培養48小時後菌體生長及膽固醇氧化生產之影響----------------------------------------------
49
圖十四 固定化R. equi No. 23於不同培養溫度下,培養48小時後菌體生長及膽固醇氧化生產之影響-------------------------------------------
50
圖十五 游離R. equi No. 23於不同振盪速率下,培養48小時後菌體生長及膽固醇氧化生產之影響----------------------------------------------
54
圖十六 固定化R. equi No. 23於不同振盪速率下,培養48小時後菌體生長及膽固醇氧化生產之影響-------------------------------------------
55
圖十七 游離及固定化R. equi No. 23於培養過程中,菌體生長與膽固醇氧化之生產之變化-------------------------------------------------------
58
圖十八 連續重複使用固定化R. equi No.23時,於各批次中菌體生長及培養液中膽固醇氧化之活性-------------------------------------------
60
表 次
頁次
表一 選擇固定化包材及方法需考量之因素------------------------------------- 15

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