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研究生:陳律安
研究生(外文):CHEN, LYU-AN
論文名稱:利用篩選之菌株生產紅色色素及其特性之研究
論文名稱(外文):Study on Production and Characterization of Red Pigments From The Isolated Bacterial Strains.
指導教授:吳建一
口試委員:顏裕鴻賴奇厚
口試日期:2016-07-26
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
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:2016
畢業學年度:104
語文別:中文
論文頁數:205
中文關鍵詞:Serratia屬紅色色素穩定性
外文關鍵詞:Serratia genusred pigmentstability
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本研究從昆蟲中分離出具有生產紅色色素的能力之細菌菌株Wu-R1和Wu-R2,16S rDNA定序結果顯示Wu-R1和Wu-R2菌株皆屬於Serratia屬,為了提高紅色色素的產量進行培養基成分及培養條件之優化探討,將Wu R1和Wu R2分別命名為Serratia sp. Wu R1和Serratia marcescens Wu R2,Serratia sp. Wu R1的最佳培養基成分為3 g/L xylose、10 g/L peptone、10 g/L NaCl和0.066 M Mg2+,Serratia marcescens Wu R2的最佳培養基成分為3 g/L xylose、5 g/L peptone、10 g/L NaCl和0.13 M Fe2+,用於生產紅色色素之最佳培養條件為0 rpm、pH 7和25℃且Serratia sp. Wu R1和Serratia marcescens Wu R2最大的紅色色素產量分別為0.16和0.14 g/L。
疏水性紅色色素使用HCl-ethanol萃取,將Wu-R1和Wu-R2萃取出的紅色色素進行LC-MS、FT-IR光譜、1 H NMR及 13 C NMR等分析,結果顯示兩株菌株生產的紅色色素皆為靈菌紅素,其分子量為324 Da,接著對紅色色素進行溫度、光照及pH值之穩定性研究,利用紅色色素濃度和L, a, b值評估紅色色素之降解特性,最後再對於紅色色素之抗氧化活性進行評估。

Bacterial strain Wu R1 and Wu R2, with the ability to produce red pigment, were isolated from the insects. The 16S rDNA gene sequencing revealed that the strain was placed in the Serratia genus. Wu R1 and Wu R2 were called Serratia sp. Wu R1 and Serratia marcescens Wu R2, respectively. The optimum medium composition was 3 g/L xylose, 10 g/L peptone, 10 g/L NaCl and 0.066 M Mg2+ from Serratia sp. Wu R1. The optimum medium composition was 3 g/L xylose, 5 g/L peptone, 10 g/L NaCl and 0.13 M Fe2+ from Serratia marcescens Wu R2. The optimum culture conditions were 0 rpm, pH 7 and 25 °C for the production of red pigment and the most production of red pigment from Serratia sp. Wu R1 and Serratia marcescens Wu R2 was 0.16 and 0.14 g/L, respectively.
The water-insoluble red pigment was extracted using HCl-ethanol. Mass spectrometry, FT-IR spectroscopy, 1H NMR and 13C NMR analysis revealed that the red pigment was prodigiosin, with molecular weights of 324 Da. The stability of red pigment was studied at temperature, pH and illumination. The degradation of pigments was evaluated by measuring red pigment concentration and the L, a, and b values. Finally, antioxidation activity of red pigment was estimated.

封面內頁
簽名頁
中文摘要 iii
ABSTRACT iv
誌謝 v
目錄 vi
圖目錄 xii
表目錄 xx

1. 前言 1
2. 文獻回顧 3
2.1天然色素來源 3
2.1.1微生物 3
2.1.2植物 6
2.1.3動物 6
2.2常見的微生物生產之紅色色素 6
2.2.1蝦青素(Astaxanthin) 6
2.2.2 β-胡蘿蔔素(β-carotene) 7
2.2.3靈菌紅素(Prodigiosin) 8
2.3 Serratia sp.簡介 8
2.3.1 Serratia sp.之特性 8
2.3.2 Serratia sp. 菌落型態與顏色 10
2.3.3 Serratia sp.生產之色素 12
2.4影響微生物生產色素的因子 17
2.4.1碳源 17
2.4.2氮源 18
2.4.3環境因子 18
2.5微生物色素的應用 20
2.5.1食品工業 20
2.5.2製藥工業 24
2.5.3紡織工業 26
2.5.4其他應用 27
3. 材料與方法 29
3.1 實驗材料 29
3.1.1 實驗藥品 29
3.1.2 儀器設備 30
3.2 菌株鑑定與培養 31
3.2.1 菌株來源 31
3.2.2 篩選菌株之鑑定 31
3.2.3菌株保存 34
3.2.4菌株活化 35
3.2.5色素生產培養 35
3.3培養基組成探討 35
3.3.1碳源種類 36
3.3.2碳源濃度 36
3.3.3氮源種類 37
3.3.4氮源濃度 37
3.4環境因子 38
3.4.1初始pH 38
3.4.2培養溫度 38
3.4.3轉速 38
3.4.4光源 39
3.4.5鹽度 39
3.5額外添加 39
3.5.1金屬離子種類 39
3.5.2金屬離子濃度 40
3.6發酵槽實驗 40
3.6.1攪拌速度 41
3.6.2曝氣量 42
3.7色素萃取條件探討 42
3.7.1萃取溶劑種類對色素吸收波長及顏色的影響 42
3.7.2萃取溶劑濃度對色素吸收波長及顏色的影響 42
3.8分析方法 43
3.8.1醣類分析-酚硫酸法 43
3.8.2氨氮分析-靛酚比色法 43
3.8.3色素分析 44
3.9紅色色素分離與純化 46
3.10紅色色素結構分析 47
3.10.1傅立葉傳換紅外光線光譜(Fourier transform infrared spectroscopy, FT-IR)分析 47
3.10.2液相層析串聯質譜儀(Liquid Chromatograph Tandem Mass Spectrometer , LC-MS)分析 47
3.10.3核磁共振(Nuclear Magnetic Resonance, NMR)分析 47
3.11紅色色素穩定性分析 48
3.11.1熱穩定性 48
3.11.2光穩定性 48
3.11.3 pH穩定性 48
3.12紅色色素之抗氧化分析 48
3.12.1 DPPH (1,1-diphenyl-2-picrylhydrazyl )自由基 清除能力分析 48
3.12.2 ABTS+陽離子自由基清除能力 49
3.12.3 亞鐵螯合能力 49
3.12.4還原力分析 49
3.12.5總酚含量分析 50
3.12.6類黃酮含量分析 50
3.12.7超氧化物歧化酶(Superoxide dismutase, SOD) 活性分析 51
3.13胞外酪胺酸酶(tyrosinase)分析 51
4. 結果與討論 53
4.1 生產紅色色素之菌株序列鑑定 53
4.2培養基組成對Serratia sp.菌株生長及紅色色素產量 之影響 57
4.2.1碳源種類對Serratia sp.菌株生長及紅色色素產量 之影響 57
4.2.2 Xylose濃度對Serratia sp.菌株生長及紅色色素 產量之影響 63
4.2.3氮源種類對Serratia sp.菌株生長及紅色色素產量 之影響 68
4.2.4 Peptone濃度對Serratia sp.菌株生長及紅色色素 產量之影響 73
4.3環境因子對Serratia sp.菌株生長及紅色色素產量 之影響 79
4.3.1初始pH對Serratia sp.菌株生長及紅色色素產量 之影響 79
4.3.2培養溫度對Serratia sp.菌株生長及紅色色素產量 之影響 85
4.3.3轉速對Serratia sp.菌株生長及紅色色素產量 之影響 91
4.3.4光源對Serratia sp.菌株生長及紅色色素產量 之影響 96
4.3.5鹽度對Serratia sp.菌株生長及紅色色素產量 之影響 102
4.4額外添加對Serratia sp.菌株生長及紅色色素產量 之影響 108
4.4.1金屬離子種類對Serratia sp.菌株生長及紅色色素 產量之影響 108
4.4.2金屬離子濃度對Serratia sp.菌株生長及紅色色素 產量之影響 114
4.5發酵槽實驗 120
4.5.1攪拌速度對Serratia sp.菌株生長及紅色色素產量 之影響 120
4.5.2曝氣量對Serratia sp.菌株生長及紅色色素產量 之影響 126
4.6紅色色素萃取條件探討 132
4.6.1萃取溶劑種類對紅色色素吸收波長及顏色的影響 132
4.6.2萃取溶劑濃度對紅色色素吸收波長及顏色的影響 136
4.7紅色色素結構分析 143
4.7.1傅立葉傳換紅外光線光譜(Fourier transform infrared spectroscopy, FT-IR)分析 143
4.7.2液相層析串聯質譜儀(Liquid Chromatograph Tandem Mass Spectrometer , LC-MS)分析 145
4.7.3核磁共振(Nuclear Magnetic Resonance, NMR)分析 148
4.8紅色色素萃取液之抗氧化分析 152
4.8.1紅色色素萃取液對DPPH自由基清除能力、 ABTS+陽離子自由基能力、亞鐵離子螯合能力 和還原力分析 152
4.8.2紅色色素萃取液中總酚、類黃酮含量和超氧化 物歧化酶(Superoxide dismutase, SOD)活性分析 157
4.9紅色色素萃取液之細胞外酪胺酸酶活性分析 161
5. 結論 163
參考文獻 166


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