臺灣博碩士論文加值系統

本研究利用反應曲面的實驗設計以獲得培養大量Pichia fermentans L-5菌體細胞之最佳培養組成為12.6 g/l 酵母抽出物及39 g/l 葡萄糖；在30℃下200rpm振盪培養進行Pichia fermentans L-5菌體轉化葡萄糖及苯丙胺酸生成乙酸苯乙酯之影響因子測試。結果發現以培養12小時（生長對數期中期）的菌體之轉化能力最強；菌量測試上發現2.4 ~ 4.8 c.d.w. g/l 的菌體濃度為適量；反應的基質濃度測試中發現27 g/l 葡萄糖及900 mg/l 苯丙胺酸為最適反應基質濃度；轉化液初始pH值為3~10時，對乙酸苯乙酯之生成無顯著影響；高通氣量則有助於轉化生成乙酸苯乙酯；轉化溫度為37.5℃時，菌體轉化生成乙酸苯乙酯之速率最快，前六小時可達70 mg l-1h-1，而於轉化8小時後即可獲得最高乙酸苯乙酯產量，約474 mg/l；於較適之轉化操作條件下，以苯丙胺酸及葡萄糖，進行反應曲面試驗設計探討，求得轉化基質之最適濃度組成為765 mg/l苯丙胺酸及20.4 g/l葡萄糖，乙酸苯乙酯之理論產量為467 mg/l，重複實驗轉化8小時後，乙酸苯乙酯生成實際值為479、484及469 mg/l。

The optimal composition of medium, 39 g/l glucose and 12.6 g/l, to produce maximum cell mass of Pihica fermentans L-5 was obtained with response surface methodology. Factors affecting bioconversion of phenylalanine to 2-phenylethyl acetate by Pihica fermentans L-5 cultivated at 30℃and 200 rpm were also investigated. The cells harvested from mid-exponential growth phase （12-hr cultivation） produced higher concentrations of 2-phenylethyl acetate. The optimum cell concentrations for production of higher concentration of 2-phenylethyl acetate was ranged from 2.4 to 4.8 c.d.w. g/l. The optimal concentration of glucose and phenylalanine for production higher concentration of 2-phenylethyl acetate were 27 g/l and 900 mg/l, respectively. The initial pH of medium, ranged from 3 to 10, showed no significant difference in 2-phenylethyl acetate production. Increase in the aeration of the medium resulted in the enhancement of production of 2-phenylethyl acetate. When cultivation temperature was at 37.5℃, the maximum productivity of 2-phenylethyl acetate 70 mg l-1h-1, was found after 6 hr of cultivation. The maximum production of 2-phenylethyl acetate 474 mg/l , was obtained after 8 hr of cultivation at 37.5℃. An optimum composition of conversion medium, i.e. 765 mg/l phenylalanine and 20.4 g/l glucose, was obtained with response surface methodology under optimum operation condition. The theoretical value of 2-phenylethyl acetate was 467 mg/l. The experimental values of 2-phenylethyl acetate produced by Pihica fermentans L-5 were 484 479 and 469 mg/l.

目錄
中文摘要
英文摘要
壹、前言………………………………………………………………1
貳、文獻整理…………………………………………………………2
(一) 食品香料之定義及分類…………………………………… 2
(二) 微生物生產香料之模式……………………………………4
1. 微生物生合成(de novo synthesis)………………………..4
2. 生物轉化(biotransformation/bioconversion)……………………5
(三) 以生物轉化法生成之芳香物質……………………………….10
1. 甲基酮類（Methyl Ketones）………………………………….10
2. 內酯類（Lactones）……………………………………………10
3. 烯類衍生物（Terpenes）……………………………………13
4. 酯類（esters）…………………………………………………..14
(四) 苯丙胺酸轉化芳香之探討……………………….……………16
1. 苯丙胺酸為芳香物之前驅物…………………………………..16
2. 轉化苯丙胺酸生成乙酸苯乙酯之探討………………………..19
3. 生物轉化系統之影響因子……………………………………..19
(1) 菌種選擇……………………………………………………..19
(2) 碳源及氨基酸的選擇……………………………………22
(3) 菌體生理期之影響…..………………………………………23
(4) 基質濃度……………………..………………………………24
(5) 轉化溫度、pH值……………………………………………25
(6) 通氣量……..…………………………………………………26
4. 乙酸苯乙酯與乙酸苯乙醇之應用……………………………..26
參、實驗材料與方法…………………………………………………..28
一、實驗材料…………………………………………………………..28
（一） 菌種……………………………………………………………28
（二） 培養基與藥品…………………………………………………28
二、實驗方法………………………………………………………….28
（一） 菌種保存與活化………………………………………………28
（二） 利用二因子反應曲面法尋求P. fermentans L-5 大量細胞之
最適培養液組成………………………………………………29
（三） P. fermentans L-5菌體於最適組成培養液下的生長探討…33
（四） P. fermentans L-5細胞懸浮液之備製………………………..33
（五） P. fermentans L-5細胞轉化生成乙酸苯乙酯及乙酸苯乙醇之
因子探討……………………………………………………..33
1. 菌體生長期之影響試驗………………………………………34
2. 菌體濃度對轉化的影響試驗…………………………………34
3. 轉化液培養組成之影響試驗…………………………………34
(1) 葡萄糖濃度之影響試驗……………………………………..34
(2) 苯丙胺酸濃度之影響試驗…………………………………..35
4. 轉化操作條件之影響試驗……………………………………35
(1) 轉化液之pH初值之影響試驗………………………………35
(2) 轉化溫度之影響試驗………………………………………..35
(3) 通氣量之影響試驗…………………………………………..35
5. 利用二因子反應曲面法尋求最適之轉化培養液組成………36
6. P. fermentans L-5菌體於最適轉化培養液下的轉化試驗……37
（六） 分析方法………………………………………………………37
1. 菌數測定………………………………………………………37
2. 活菌數測定…………………………………………………….37
3. 菌體乾重……………………………………………………….37
4. pH測定…………………………………………………………39
5. 葡萄糖含量測定……………………………………………….39
6. 苯丙胺酸、乙酸苯乙酯及乙酸苯乙醇之含量測定………….39
肆、結果與討論……………………………………………………….41
（一） 利用二因子反應曲面法尋求P. fermentans L-5 大量細胞之
最適培養液組成………………………………………………41
（二） P. fermentans L-5菌體於最適培養液下的生長情形………..46
（三） P. fermentans L-5細胞轉化生成乙酸苯乙酯及乙酸苯乙醇
之因子探討……………………………………………………49
1. 菌體生長期之影響試驗………………………………………..49
2. 菌體濃度對轉化的影響探討…………………………………..54
3. 轉化液培養組成之影響試驗…………………………………..59
(1) 葡萄糖濃度對轉化的影響試驗……………………………..59
(2) 苯丙酸濃度對轉化的影響試驗……………………………..65
4. 轉化操作條件探討……………………………………………..69
(1) 轉化液之pH初值之影響試驗………………………………71
(2) 轉化溫度之影響試驗…………………………………..73
(3) 通氣量之影響試驗…………………………………………..78
(4) 利用二因子反應曲面法尋求最適之轉化培養液組成……..82
(5) P. fermentans L-5菌體於最適轉化培養液下的轉化試驗….94
伍、結論………………………………………………………………97
陸、參考文獻…………………………………………………………99
柒、附錄………………………………………………………………107
圖次
頁次
圖一 利用Penicillium roqueforti孢子氧化脂肪酸轉換成甲基酮….11
圖二 利用Yarroowia lipolytica 細胞轉換ricionoeic acid生成γ-
decalactone………………………………………………………12
圖三 利用Pseudomonas sp.L降解 (+)-limonene之代謝路徑……..15
圖四 以生物技術生成benzaldehyde 及2-phenylethyl alcohol 之路
徑推測……………………………………………………………17
圖五 Ehrich 代謝路路徑……………………………………………..20
圖六 推測phenylalanine生物轉化生成2-phenylethyl acetate之路
徑…………………………………………………………………21
圖七Glucose及yeast extract濃度對培養24小時P. fermentans L-5
菌體濃度（OD600）之反應曲面圖及等高線圖………………..47
圖八 P. fermentans L-5於最適培養基下之生長，培養液pH變化
及葡萄糖消耗情形……………………………………………..48
圖九 不同生長期之P. fermentans L-5於轉化過程中苯丙胺酸及葡
萄糖消耗和乙酸苯乙酯及乙酸苯乙醇產生之情形……………51
圖十 不同P. fermentans L-5菌體濃度，於轉化過程中苯丙胺酸及
葡萄糖消耗和乙酸苯乙酯及乙酸苯乙醇產生之情形…………55
圖十一 不同葡萄糖濃度，對P. fermentans L-5菌體於轉化過程
中，苯丙胺酸和酸葡萄糖和乙酸苯乙酯及乙酸苯乙醇產
生之情形………………………………………………………60
圖十二 不同苯丙胺酸濃度，對P. fermentans L-5菌體於轉化過
程中，苯丙胺酸和酸葡萄糖消耗和乙酸苯乙酯及乙酸苯
乙醇產生之情形………………………………………………67
圖十三 不同葡萄糖濃度，對P. fermentans L-5菌體於轉化過程中，
苯丙胺酸和酸葡萄糖和乙酸苯乙酯及乙酸苯乙醇產生之情
形……………………………………………………………..72
圖十四 不同培養溫度，對P. fermentans L-5菌體於轉化過程中，
苯丙胺酸和酸葡萄糖消耗和乙酸苯乙酯及乙酸苯乙醇產生
之情形……………………………………………………….76
圖十五 不同培養通氣量，對P. fermentans L-5菌體於轉化過程
中，苯丙胺酸和酸葡萄糖消耗和乙酸苯乙酯及乙酸苯乙
醇產生之情形…………………………………………………81
圖十六 培養4小時後苯丙胺酸濃度與葡萄糖濃度對於乙 酸苯乙
酯生成量之等高線圖…………………………………………85
圖十七 培養8小時後苯丙胺酸濃度與葡萄糖濃度對於乙酸苯乙
酯生成量之等高線圖…………………………………………93
圖十八 最適轉化培養組成下，P. fermentans L-5菌體生長、葡萄
糖和苯丙胺酸消耗及乙酸苯乙酯和乙酸苯乙醇生成之情
形…………………………………………………………….95
表次
頁次
表一 微生物以生合成方式生成之芳香物質…………………………6
表二 利用微生物細胞或或酵素之生物轉化反應……………………7
表三 利用lipase於胞外合成酯類芳香物質…………………………18
表四 生成菌體細胞之兩因子五變級之十三組試驗中心組成設計…31
表五 轉化液組成之二因子五變級之十三組試驗中心組成設計……38
表六 生成菌體細胞中心組成設計之試驗結果………………………43
表七 反應曲面試驗設計之P. fermentans L-5菌量（OD600）之變異
性分析……………………………………………………………44
表八 菌體生成量之複迴歸係數………………………………………45
表九 不同生長期下，P. fermentants L-5之生長，培養液pH終值
及乙酸苯乙酯與乙酸苯乙醇之產生……………………………53
表十 不同P. fermentants L-5細胞濃下，P. fermentants L-5之生長
，培養液pH終值及乙酸苯乙酯與乙酸苯乙醇之產生……….58
表十一 不同葡萄糖濃度下，P. fermentants L-5之生長，培養液
pH終值及乙酸苯乙酯與乙酸苯乙醇之產生………………..64
表十二 不同苯丙胺酸濃度下，P. fermentants L-5之生長，培養液
pH終值及乙酸苯乙酯與乙酸苯乙醇之產生………………..70
表十三 不同初始 pH值下，P. fermentants L-5之生長，培養液
pH終值及乙酸苯乙酯與乙酸苯乙醇之產生………………..74
表十四 不同溫度下P. fermentants L-5之生長，培養液pH終值
及乙酸苯乙酯與乙酸苯乙醇之產生…………………………79
表十五 不同通氣量下，P. fermentants L-5之生長，培養液pH
終值及乙酸苯乙酯與乙酸苯乙醇之產生……………………83
表十六 轉化液組成之中心組成設計之試驗結果……………………86
表十七 反應曲面試驗設計之生成乙酸苯乙酯變異性分析…………87
表十八 培養4小時後乙酸苯乙酯生成量回歸模式之複迴歸係數…88
表十九 反應曲面試驗設計之生成乙酸苯乙酯變異性分析…………90
表二十 培養8小時後乙酸苯乙酯生成量回歸模式之複迴歸係數…92

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