臺灣博碩士論文加值系統

蝦紅素(Astaxanthin)是一種萜烯類不飽和化合物，在600多種類胡蘿蔔素中極為昂貴。近年來已經有多份研究報告指出蝦紅素具有提升免疫機能、抑制腫瘤細胞的功能。酵母菌生產蝦紅素之液態培養乃目前各大廠商主要研究的培養方式，可在短時間、有限空間下生產大量酵母菌蝦紅素，極具有商業經濟發展能力。
根據本研究發酵結果發現，Xanthophyllomyces dendrorhous酵母菌所產的蝦紅素的量較Phaffia rhodozyma酵母菌所產的蝦紅素要多，接著使用發酵槽做pH的調控，可以比較出有調控pH值產的蝦紅素量較沒控制pH的蝦紅素產量還要高，在發酵槽養殖60小時Phaffia rhodozyma酵母菌所產的蝦紅素產量為1.87 mg/g DCW，而Xanthophyllomyces dendrorhous酵母菌所產的蝦紅素產量則是2.34 mg/g DCW，Xanthophyllomyces dendrorhous酵母菌生產蝦紅素有調控pH值和沒調pH值的產量分別為0.4713 g-biomass/ g-glucose以及0.4127 g-biomass/ g-glucose。而後用四種不同萃取方法，萃取兩株不同酵母菌所產之蝦紅素，Phaffia rhodozyma酵母菌在四種方法的蝦紅素產量為32μg/ml、49μg/ml、0μg/ml、0μg/ml，Xanthophyllomyces dendrorhous酵母菌在四種方法的蝦紅素產量為43μg/ml、56μg/ml、0μg/ml、0μg/ml，可達到Xanthophyllomyces dendrorhous酵母菌在方法B時的蝦紅素產量最多。利用酵母菌蝦紅素對Actin基因做重組調取分析，再將蝦紅素對YM、MM、MB三種不同培養基進行培養和蝦紅素的含量比較，了解MM為最佳培養基後做MM培養基的最適碳源濃度，在MM培養基中酵母菌蝦紅素最適碳源濃度為50g/L，可獲得640 g/ml蝦紅素含量。
最後將蝦紅素在HPLC的鑑定下，藻類蝦紅素只有S波峰出現，酵母菌蝦紅素則只有R波峰出現，質譜分析儀上藻類的MASS為595.4 m/z，酵母菌的MASS為593 m/z。在抗氧化能力的測試，用DPPH後知道蝦紅素的抗氧化能力為4ppm，而beta胡蘿蔔素的抗氧化能力只在3ppm，明顯的蝦紅素抗氧化能力較強。

關鍵字：蝦紅素、藻類、酵母菌

Astaxanthin is a type of unsaturated compound terpenoids which is highly valuable among over 600 types of carotenes. There have been multiple researches that report on the functions of Astaxanthin in enhancing immune system and inhibit tumor cells. The liquid culture of Astaxanthin produced from yeasts is the primary culture method used in research conducted by major companies for that a massive amount of yeast-based Astaxanthin produced within a short time and limited space has a high potential in the commercial and economic development.
The fermentation results of the study show that the amount of Astaxanthin produced from Xanthophyllomyces dendrorhous yeast was more than the amount produced from Phaffia rhodozyma yeast. Next, by using a fermentor as pH control, the amount of Astaxanthin produced with pH control was comparatively higher than the amount of Astaxanthin produced without pH controlled. The output of Astaxanthin produced from Phaffia rhodozyma yeast under 60 hours of fermentor culture was 1.87 mg/g DCW, while the output of Astaxanthin produced from Xanthophyllomyces dendrorhous yeast was 2.34 mg/g DCW. The outputs for Astaxanthin produced from Xanthophyllomyces dendrorhous yeast with pH value controlled and without were 0.4713 g-biomass/ g-glucose and 0.4127 g-biomass/ g-glucose, respectively. Later, we used four different extraction methods to extract the Astaxanthin produced from two strains of different yeasts, whereas the outputs of Astaxanthin produced from Phaffia rhodozyma yeast through the methods were 32μg/ml, 49μg/ml, 0μg/ml, and 0μg/ml, respectively. The outputs of Astaxanthin produced from Xanthophyllomyces dendrorhous yeast through the methods were 43μg/ml, 56μg/ml, 0μg/ml, and 0μg/ml respectively. The maximum output of Astaxanthin was attained from Xanthophyllomyces dendrorhous yeast by using Method B. The Astaxanthin produced from yeasts were recombined, trapped and analyzed with Actin genes, followed by development culture on the three different mediums, YM, MM and MB, in addition to comparing the content of Astaxanthin. It was discovered that MM was the best medium and was used as the optimal carbon source concentration for MM medium. The optimal carbon source concentration for the Astaxanthin of yeasts in MM medium was 50g/L, yielding a content of 640 g/ml Astaxanthin.
Finally, the Astaxanthin underwent HPLC identification and it was discovered that algae-based Astaxanthin only showed S wave while yeast-based Astaxanthin only showed R wave. The MASS of algae on the spectrum analyzer was 595.4 m/z while the MASS of yeast was 593 m/z. During the test for anti-oxidation, the anti-oxidation of Astaxanthin using DPPH was 4ppm while the anti-oxidation of Beta carotenes was only 3ppm, indicating significantly stronger anti-oxidation of Astaxanthin.

Keywords: Astaxanthin, Algae, Yeast

明志科技大學碩士學位論文指導教授推薦書 ......i
明志科技大學碩士學位論文口試委員審定書 ......ii
明志科技大學學位論文授權書......iii
誌謝......iv
中文摘要......v
英文摘要......vii
目錄......ix
第一章 緒論......1
第二章 文獻回顧......2
2-1蝦紅素介紹......2
2-1-1蝦紅素的性質結構......3
2-1-2蝦紅素存在型態......4
2-2蝦紅素功能性......4
2-2-1抗氧化與自由基之間的關係......4
2-2-2蝦紅素為抗氧化劑......5
2-3蝦紅素與其他疾病......6
2-3-1降低紫外線對皮膚的傷害......7
2-3-2預防心血管疾病......8
2-3-3降低對視網膜的傷害，預防白內障......9
2-3-4防治發炎......10
2-3-5預防腦缺血......10
2-3-6抗癌......10
2-4蝦紅素的生產來源......11
2-4-1從水産加工廢棄物中提取蝦紅素......11
2-4-2雨生紅球藻生產蝦紅素......13
2-4-3紅酵母菌生產蝦紅素......16
2-5提高酵母菌蝦紅素產量的方法......18
2-5-1氧和葡萄糖對Phaffia rhodozyma酵母菌蝦紅素的影響......19
2-5-2溫度和pH對酵母菌蝦紅素的影響......19
2-5-3 光照和抗生素對酵母菌蝦紅素的影響......20
2-6蝦紅素的萃取......20
2-6-1化學修飾合成法......20
2-6-2有機溶劑的萃取......21
2-6-3 CO2超臨界流體萃取......23
第三章 實驗材料與方法......25
3-1儀器......25
3-2實驗材料......26
3-2-1菌種來源......26
3-2-2藥品......26
3-3實驗架構......29
3-4實驗方法......30
3-4-1菌種培養與保存方法......30
3-4-1-1酵母菌之保存......30
3-4-1-2酵母菌放大及保存......30
3-4-2發酵培養......31
3-4-2-1裝配發酵槽......31
3-4-2-2接菌培養......31
3-4-2-3酵母菌蝦紅素發酵培養方法......32
3-4-3蝦紅素之分析......33
3-4-3-1 Phaffia rhodozyma蝦紅素定量取樣......33
3-4-3-2 Xanthophyllomyces dendrorhous蝦紅素定量取樣......34
3-4-3-3三種不同培養基對Phaffia rhodozyma蝦紅素的含量......34
3-4-4蝦紅素四種方法含量測定......35
3-4-4-1方法一.荊州天然蝦青素有限公司的方法......35
3-4-4-2方法二.美國Cyanotech公司的測定方法......35
3-4-4-3方法三.氯仿一乙醇混合溶液提取方式 ......36
3-4-4-4方法四.......36
3-4-5 DPPH蝦紅素抗氧化測定......36
3-4-5-1 DPPH法測定抗氧化原理......36
3-4-5-2 DPPH之測定......37
3-4-6 FRAP (ferric reducing antioxidant power)分析......37
3-4-6-1 FRAP法測定抗氧化力之原理......37
3-4-6-2 FRAP之測定......38
第四章 結果與討論......39
4-1酵母菌生產蝦紅素......39
4-1-1 Xanthophyllomyces dendrorhous蝦紅素之生產......39
4-1-2 Phaffia rhodozyma蝦紅素之生產......40
4-2最適培養基之碳源濃度......41
4-3蝦紅素最適化之萃取......42
4-3-1蝦紅素四種萃取方法之比較......42
4-4蝦紅素之鑑定......45
4-4-1蝦紅素之光學異構體......45
4-4-2蝦紅素在質譜儀之表現......45
4-5蝦紅素抗氧化能力之最適化......47
4-5-1 DPPH對酵母蝦紅素測定......47
4-5-2 FRAP對酵母蝦紅素測定......48
第五章 結論與未來展望......49
附錄......51
附錄一 實驗常用藥品之配置......51
附錄二 抽取DNA所需之藥品配置......53
參考文獻......54

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