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研究生： 林興泉
Rhesa - Pramudita Utomo
論文名稱： Wet Process of Lutein Extraction from Chlorella vulgaris
Wet Process of Lutein Extraction from Chlorella vulgaris
指導教授： 李篤中
Duu-Jong Lee
口試委員: 劉志成
Jhy-Chern Liu
Christopher George Whiteley
Christopher George Whiteley
學位類別： 碩士
Master
系所名稱： 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年： 2013
畢業學年度： 101
語文別： 英文
論文頁數： 88
中文關鍵詞： 葉黃素 、小球藻 、濕式製程 、超聲波 、微波 、甲殼素 、聚氯化鋁
外文關鍵詞： Lutein, Chlorella vulgaris, wet process, ultrasonic, microwave irradiation, chitosan, PACl
相關次數： 點閱：369  下載：14 
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    • 保健在現代社會是一個非常重要的議題, 也是人們致力的目標, 但隨著老化, 眼睛會因累積了光氧化物質(photooxidative), 進而導致老年性黃斑部病變 (AMD)
    在2001, 美國國家眼科研究所(NEI) 證實了葉黃素可以有效延緩AMD的發生.
    目前葉黃素主要由金盞花的花辮中萃取, 但產率僅有 0.03 %. 微藻由於有較高葉黃素的含量, 有取代金盞花潛力, 從微藻中生產葉黃素需要像是冷凍乾燥等較先進的製程, 在本論文中, 本人試圖利用濕式製程取代冷凍乾燥的需求並評估此製程對葉黃素產率的影響.
    使用Bold’s Basal Medium為培養液, 在25oC與光照度1945流明下, 每兩天觀察葉黃素在微藻Chlorella vulgaris中的累積變化, 發現到第六天時微藻具有最高的葉黃素含量. 在濕式製程中, 引入細胞破碎法能提升葉黃素萃取的成效. 在本論文中, 最佳細胞破碎方式為微波(Microwave irradiation, 1.5 min, 100 W), 產率可達92.92±2.30%, 相較於超聲波細胞破碎法的87.48±1.71% (20 kHz ultrasonic, 2 min, 130 W) 與85.20±2.25% (42 kHz ultrasonic, 0.5 min, 135 W). 在微藻收集中, 使用甲殼素與聚氯化鋁的混凝技術, 再藉由在 1 bar與2 bar不同壓力下的膜過濾, 收集微藻絮凝物. 研究中發現, 在混凝劑的添加下, 會降低葉黃素萃取的產量, 其中甲殼素的影響較聚氯化鋁顯著. 粗萃取物在HPLC的分析下, 在4.5-5分鐘時可得純度為98.82 ± 0.02%的葉黃素.

    Health always becomes top priority of human kind. People already made many efforts to achieve it, but all people cannot stay healthy forever. It is because of ageing. With time, the aging eye accumulates more photooxidative and leads to age-related macular degeneration (AMD). In 2001, the National Eye Institute (NEI) confirmed that lutein can delay the progression of late stage AMD.
    Currently, lutein is obtained from the petals of marigold which has low yield 0.03%. Microalgal biomass can substitute the marigold to give better lutein yield. The production of lutein from microalgae demands more advanced technology such as lyophilization process by using freeze dyer. In this research, the author attempted to eliminate the need of freeze drying the microalgal biomass prior to the extraction processes and evaluate its effect toward lutein yield. The author called that elimination as wet process.
    The accumulation of lutein in Chlorella vulgaris per 2 day was observed. It was found that the 6th cultivation (25oC Bold’s Basal Medium with light intensity 1945 lux) gave the highest amount of lutein. The usage of cell disruption method in wet process can increase well the performance of lutein extraction. Microwave irradiation (1.5 min, 100 W) gave the best performance i.e. 92.92±2.30% followed by 20 kHz ultrasonic (2 min, 130 W) i.e. 87.48±1.71% and 42 kHz ultrasonic (0.5 min, 135 W) i.e. 85.20±2.25%. Coagulation technique by using chitosan and/or PACl (10% w/w Al2O3) in Chlorella vulgaris isolation step was also examined. Membrane filtration at 1 bar and 2 bar were used to separate the coagulated Chlorella vulgaris. It was found that PACl decreased the yield of lutein in 1 bar and 2 bar membrane filtration, chitosan decreased the yield of lutein in greater amount compared to PACl, and 2 bar system had less lutein yield compared to 1 bar. The highest amount of pure lutein (98.82 ± 0.02%) from wet processed crude lutein was obtained on 4.5-5.0 minute by using HPLC.

    ABSTRACT i 摘要 ii ACKNOWLEDGEMENT iii CONTENTS iv LIST OF TABLES vi LIST OF FIGURES vii CHAPTER 1. INTRODUCTION 1-1 1.1. Background 1-1 1.2. Objective 1-2 1.3. Research plan 1-3 CHAPTER 2. LITERATURE REVIEW 2-1 2.1. Carotenoids 2-1 2.2. Chemical and biological synthesis of carotenoids 2-2 2.3. Lutein 2-4 2.4. Microalgae as sources of carotenoids 2-6 2.5. Methods of lutein extraction 2-9 2.6. Anatomy of retina 2-14 2.6.1. Photoreceptors 2-14 2.6.2. The retinal pigment epithelium (RPE), bruch’s membrane and choriocapillaris 2-15 2.6.3. Macula 2-16 2.7. Age-related macular degeneration (AMD) 2-16 CHAPTER 3. MATERIALS AND METHODS 3-1 3.1. Chemical and materials 3-1 3.2. Equipment 3-3 3.3. Microalgae and culture medium 3-4 3.4. Analytical methods 3-5 3.4.1. Determination of maximum absorbance of lutein via UV/VIS spectrophotometer 3-5 3.4.2. Calibration curve of standard lutein 3-6 3.4.3. Determination of cell concentration 3-8 3.4.4. Determination of lutein content in Chlorella vulgaris 3-9 3.4.5. Evaluation of 20kHz ultrasonic treatment toward lutein yield in wet process 3-10 3.4.6. Evaluation of 40kHz ultrasonic treatment toward lutein yield in wet process 3-10 3.4.7. Evaluation of microwave treatment toward lutein yield in wet process 3-11 3.4.8. Evaluation of coagulation of Chlorella vulgaris toward lutein yield in wet process 3-11 3.4.9. Evaluation of extracellular organic matter (EOM) of Chlorella vulgaris toward lutein yield in wet process 3-12 3.4.10. Purification of crude lutein from wet process by using HPLC 3-14 3.4.11. Verification of lutein in the analyte by using internal standard in HPLC analysis 3-14 CHAPTER 4. RESULT AND DISCUSSION 4-1 4.1. Characteristic of Chlorella vulgaris 4-1 4.2. Determination of lutein content in Chlorella vulgaris 4-4 4.3. Evaluation of different cell wall disruption methods toward lutein yield in wet process 4-5 4.4. Evaluation of coagulation of Chlorella vulgaris toward lutein yield in wet process 4-12 4.5. Evaluation of extracellular organic matter (EOM) of Chlorella vulgaris toward lutein yield in wet process 4-24 4.6. Purification of crude lutein from wet process by using HPLC 4-24 4.7. Verification of lutein in the analyte by using internal standard in HPLC analysis 4-24 CHAPTER 5. CONCLUSIONS 5-1 REFERENCES R-1 APPENDIX A-1

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