臺灣博碩士論文加值系統

　　黏質沙雷氏菌（Serratia marcescens）屬於腸內桿菌科（Enterobacteriaceae），為革蘭氏陰性菌，常見於土壤及水中等環境中，為靈菌紅素的主要生產者。靈菌紅素是色素性二次代謝物，主要由Serratia marcescens、Vibrio psychroerythrus、Hahella chejuensis、Pseudomonas magnesiorubra 等微生物所生產。靈菌紅素具有多種生物活性作用，例如：抗癌、抗微生物、抗瘧疾、抗藻、免疫抑制等生理活性。因此，找出能夠大量生產靈菌紅素的菌株，及大量生產靈菌紅素便成為一項很重要的研究。本研究計畫選用本實驗室分離出的菌株黏質沙雷氏菌 YOR-1，並根據黏質沙雷氏菌產靈菌紅素進行基礎培養基的修改，同時針對固態培養與液態培養進行測試，由試驗結果得知固態培養 靈菌紅素的產量比液態培養高1.83倍，試驗結果也發現不同體積的液態培養的單位細胞所含色素量，其培養體積越大，則色素產量便會下降，而前人研究皆以液態培養為主，鮮少有固態培養研究發表。固態培養不受體積大小、接種量及含氧量等因素而影響產量，且產量高出許多，因此本實驗選用固態培養方式生產靈菌紅素，並修改營養肉湯培養基實驗中改變蛋白腖及牛肉萃取物的濃度，再以成本效益分析得到最佳生產色素的濃度為0.4000% 蛋白腖及0.2000% 牛肉萃取物，並添加不同的氮源及碳源，來找出最適合此菌株生產靈菌紅素的營養源，實驗結果其最佳氮源及碳源分別為麥芽萃取物及果糖。本研究計畫以麥芽萃取物及果糖此兩種因子設計不同濃度以求得最適化培養基。最適化培養基果糖濃度為 0.6086%、麥芽萃取物濃度為0.0039%的條件之下為生產靈菌紅素的最佳培養基濃度。實驗結果顯示，最佳培養基濃度所得到的靈菌紅素濃度為4.2822 g/L。

Prodigiosin is one of secondary metabolites produced by Serratia marcescens, Vibrio psychroerythrus, Hahella chejuensis, Pseudomonas magnesiorubra. Prodigiosin has many bio-activities like anti-cancer ,anti-bacterial, anti-malarial, anti-algae activity and many more applications. Therefore, find the optimal condition to increase the production yield of prodigiosin is very important for industrial production. In this study, we used S. marcescens YOR-1 which could produce prodigiosin was isolated from environment. The first step, we modify the formula of the basic medium（nutrient broth）. Modified nutrient broth content the concentration of 0.4000% peptone and 0.2000% beef extract. Next, we compare the producing yield of prodigiosin in solid-state culture and liquid culture. We also compare the different in solid-state culture and liquid culture. The result indicate large culture volume in liquid culture will reduced prodigiosin production yield, but not affected in solid-state culture. Beside this, we also discovered the prodigiosin yield in solid-state culture is 1.83 times higher than the liquid culture. Therefore, in this study, we used solid-state culture methods to produce prodigiosin, and test different nitrogen source and different carbon source to used in the prodigiosin production by S.marcescens YOR-1.The highest prodigiosin production yield was observed in malt extract and fructose. The experimental design was used to optimize the medium constituents for maximal prodigiosin production yield by S. marcescens. Results showed that the optimal medium constituents were 0.0039% malt extract and 0.6086% fructose. Under this optimal composition, the prodigiosin production yield was 4.2881 g/L.

目錄
中文摘要
英文摘要
目錄………………………………………..……………………………………………..I
表目錄………………………………………..………………………………………...III
圖目錄………………………………………..………………………………………...IV
壹、前言 ………………………………………..………………………………………1
貳、材料 ……………….………………………………………………………………..6
一、實驗菌株 ………………………………………………………………………...6
二、實驗藥品 …...……………………………………………………………………6
參、培養基配方…………………………………………………………………………7
一、保存培養基配方…………………………………………………………………7
二、培養基篩選測試培養基配方……………………..………..……………………8
肆、實驗儀器及設備……………………………………………...…………………..10
伍、 實驗方法……………………………………………………...…………………..11
(一) 種菌培養 ……………………………………………………..……..……....…..11
(二) 靈菌紅素萃取與測量………………………………………...….……………….11
(三) 細胞乾重定量……………………………………………………………………12
(四) 菌種鑑定…………………………………………………………………………13
(五) 生化特性分析……………………………………………………………………14
(六) 培養基篩選………………………………………………………………………15
(七) 培養方式篩選……………………………………………………………………15
(八) 培養條件篩選……………………………………………………………………15
(九) 基礎培養基修改…………………………………………………………………16
(十) 營養源篩選………………………………………………………………………17
(十一) 最適生產條件……………………………………………………………….18
陸、結果………………...……………………………………………………………..19
一、菌株鑑定及特性分析……………….……………………..………………..…19
二、培養液篩選試驗…………………….……………………………..…………..19
三、液態培養與固態培養所得之靈菌紅素產量比較………….……..………..…19
四、不同環境因子篩選………………….…………………………………………20
五、基礎培養基修改…………………….…………………………………………20
六、營養源因子篩選…………………….…………………………………………22
七、最適化生產靈菌紅素培養基…….……………….…………………………...22
柒、討論……………………………………………………………………………….28
捌、實驗表與圖……………………………………………………………………….30
玖、參考文獻………………………………………………………………………….60
附錄、……………………………………………………………………………….....62












表目錄
表一、Serratia marcescensYOR-1之生化特性及與標準菌株比較 ………….…….30
表二、第一次最適化生產靈菌紅素培養基濃度設計…………………..……………31
表三、第一次試驗濃度設計回歸分析表 ……………………………….…………..32
表四、第一次試驗濃度設計變方分析表 ……………………………….…………..32
表五、第二次最適化生產靈菌紅素培養基濃度設計 …………….….…………….33
表六、第一次試驗濃度設計回歸分析表 ……………………………….…………..34
表七、第一次試驗濃度設計變方分析表 ……………………………………….…..34
表八、第三次最適化生產靈菌紅素培養基濃度設計 ……………………….……..35
表九、第一次試驗濃度設計回歸分析表 ……………………………….…………..37
表十、第一次試驗濃度設計變方分析表 ………………………….………………..37
表十一、最適化生產靈菌紅素之文獻整理 ……………………….……….……….38











圖目錄
圖一、靈菌紅素與其衍生物結構…………..……………….…………………………39
圖二、靈菌紅素合成路徑 …………………………………………….………………40
圖三、靈菌紅素濃度與吸光值之曲線關係 …..………….…….…………………….41
圖四、YOR-1之演化樹圖 …..…………………………..…………………………….42
圖五、不同培養液篩選測試 ……………………………………………………….....43
圖六、液態培養與固態培養所得之靈菌紅素產量比較 …………………………….44
圖七、培養條件篩選 ………………….……………………………………………....45
圖八、基礎培養基修改-固定0.4% 蛋白腖，分別添加不同濃度之牛肉萃取物……46
圖九、基礎培養基修改-成本效益分析比較圖 ………………………………...……47
圖十、基礎培養基修改-固定0.2%牛肉萃取物，分別添加不同濃度之蛋白腖……48
圖十一、基礎培養基修改-成本效益分析比較圖 ……………………………....……49
圖十二、營養源篩選-添加不同碳源篩選影響靈菌紅素生成之因子 ………………50
圖十三、營養源篩選-添加不同氮源篩選影響靈菌紅素生成之因子 ………………51
圖十四、營養源篩選-添加不同無機鹽類篩選影響靈菌紅素生成之因子…..………52
圖十五、第一次因子試驗等高線圖 ……………………………………………….....53
圖十六、第一次因子試驗交互作用圖 ………………………………………..…...…54
圖十七、第二次因子試驗等高線圖 ……………………………………………….....55
圖十八、第二次因子試驗交互作用圖 ..……………………………………………...56
圖十九、第三次因子試驗等高線圖 ………………………………………………....57
圖二十、因子試驗設計最佳反應圖 ………………………………………………....58
圖二十一、因子試驗設計最佳反應圖 ………………………….…………………....59

林芊 (2005) 由粘質沙雷氏菌生產靈菌紅素之研究。國立台灣大學農業化學研究所碩士論文

魏毓宏、蔡敏君、王莉娟、蘇怡禎 (2007) 天然抗癌藥物靈菌紅素之微生物醱酵製程開發與應用。行政院國家科學委員會補助專題研究計畫報告

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