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系統識別號 U0026-1808201516125300
論文名稱(中文) 以本土微藻生產葉黃素及其萃取及保存程序開發
論文名稱(英文) Production, extraction, and stabilization of lutein from microalgae
校院名稱 成功大學
系所名稱(中) 化學工程學系
系所名稱(英) Department of Chemical Engineering
學年度 103
學期 2
出版年 104
研究生(中文) 曾紫敏
研究生(英文) Jesisca
學號 N36025022
學位類別 碩士
語文別 英文
論文頁數 83頁
口試委員 指導教授-張嘉修
口試委員-近藤昭彥
口試委員-張鑑祥
口試委員-陳炳宏
口試委員-吳意珣
中文關鍵字 葉黃素  微藻  Chlorella sorokiniana  曝氣  醋酸鈉  半批次  超臨界流體  減壓濃縮萃取  穩定性  植物油 
英文關鍵字 lutein, microalgae  Chlorella sorokiniana  aeration  sodium acetate  semi-batch  supercritical carbon dioxide  reduced pressure extraction  stabilization  vegetable oils 
學科別分類
中文摘要 葉黃素是一種類胡蘿蔔素,存於植物、微藻和其他光合生物。葉黃素於人類視網膜裡有過濾藍光之作用,以保護眼睛免受到光之傷害。葉黃素是眾所皆知可有效降低黃斑部病變發生之保健食品,近年來已被大量地推廣使用。近幾年來,利用微藻生產葉黃素已有諸多研究並引起廣大之興趣。相對於目前商業化葉黃素之主要來源-金盞花,利用微藻生產葉黃素有許多優點,例如微藻有較高之生長速率及葉黃素含量、且微藻生長並無季節性之限制,且其養殖之人力需求較低等。但目前文獻中仍缺乏完整的微藻葉黃素下游製程之資訊。
本研究首先以自行篩選之本土藻株Chlorella sorokiniana MB-1進行葉黃素生產之探討,針對氣體種類、醋酸鈉濃度及半批次等操作因子對葉黃素含量及生產速率之影響進行探討。藻株C. sorokiniana MB-1培養之基礎條件為:BG-11培養基,硝酸鈉濃度為1 g/L,光照度150 µmol/m2/s,曝氣速率0.1 vvm。先進行氣體種類之探討,分別為曝二氧化碳(CO2)、曝空氣及不曝氣進行培養。其結果顯示,進行二氧化碳曝氣培養時,C sorokiniana MB-1可獲得較高之葉黃素含量及生產速率。接續將醋酸鈉濃度進行探討,發現最佳醋酸鈉濃度為6.0 g/L,葉黃素含量可達到6.28 mg/g,同時葉黃素生產速率也可高達3.06 mg/L/d。為了進一步提高葉黃素之含量,本研究也進行半批次操作之探討,進行不同基質置換比例之最適化探討,當基質置換比例為75%時其葉黃素之含量有較好之表現。以此條件進行八次循環之半批次操作時,其葉黃素產率可提升至5.78 mg/L/d。
接著使用超臨界流體萃取(SFE)和減壓濃縮萃取兩種方法來進行微藻葉黃素之回收。結果顯示,從SFE所獲得的葉黃素回收率只有22 %,但使用減壓濃縮萃取可得到較高的回收率。此外,相較於傳統溶劑之萃取方法,減壓濃縮萃取可在較低溫之環境下進行,故可防止葉黃素的降解,且其溶劑可循環回收利用,故能有效地減少有機溶劑的消耗。研究發現,葉黃素之回收率主要取決於以下參數:前處理,壓力,溶劑種類,萃取時間和溫度等因素。本研究使用高效能液態層析儀(HPLC)進行葉黃素回收之測試。於前處理的部分,選用高壓細胞破碎儀,其效果較球磨機更為有效。結果顯示,四氫呋喃(THF)對於葉黃素之回收率與其他溶劑相比有較好之效果。當使用高壓細胞破碎儀前處理,接著使用減壓濃縮進行萃取,溶劑種類為THF,其壓力、溫度及時間條件依序為 850 mbar、25°C及20分鐘時,葉黃素回收率可達87.0%,若須將葉黃素99.5%回收,則需延長其萃取時間至40 分鐘。雖然THF有較高之回收率,但由於乙醇相較於THF較適合使用於食品加工製程,因此進一步探討以乙醇做為溶劑進行萃取,並探討其最適化之條件。結果發現,使用乙醇作為溶劑,於壓力450 mbar、溫度35°C及時間40分鐘條件下,葉黃素的回收可達86.2%。
為維持葉黃素之穩定性,本研究探討保存於植物油中之效果,主要針對葉黃素保存於橄欖油和葵花油中之穩定性進行研究,並進行兩種保存溫度 (4℃和25℃)之效果。葉黃素樣品於特定保存時間進行分析,並以動力學分析探討葉黃素之穩定性和其半衰期。初步研究發現,使用植物油來保存葉黃素仍無法阻止葉黃素之光降解,故須把樣品儲存於暗室以增加其穩定性。實驗結果顯示,葉黃素儲存於橄欖油中之半衰期,在4℃環境下為59.1天;25℃環境下為22.4天。使用葵花油之相對應結果為4℃環境下為63.8天;25℃環境下為31.3天。其穩定性之差異推測原因為存在於兩種不同油品內之抗氧化成分含量不同所致。由上述結果可知,植物油能夠有效延長葉黃素之穩定性,明顯優於控制組 (葉黃素乙醇萃取液),其半衰期於4℃和25℃環境下僅分別為16.7天和11.1天。
英文摘要 Lutein is a type of carotenoids that can be found in plants, microalgae and other photosynthetic organisms. Lutein in human retina acts as a blue light filter to protect eyes from photochemical damage. Lutein, widely known as health supplement to reduce age-related macular degeneration (AMD) risk, has been well developed with increasing demand over the years. Lutein production from microalgae has been drawing numerous research interests over the past few years. Microalgae show many advantages as lutein source over commercial lutein source (i.e., marigold flower), such as the high growth rate, high lutein content, no seasonal limitation and less labor-demand.
In this study, lutein production from Chlorella sorokiniana MB-1 strain was examined. The effects of gas, sodium acetate concentrations, and semi-batch operation on lutein content and lutein productivity were demonstrated. First, C. sorokiniana MB-1 were grown on BG-11 medium with 1.0 g/L of sodium nitrate, light intensity of 150 µmol/m2/s and 0.1 vvm aeration rate. Carbon dioxide (CO2) and air were used as feeding gas, and another batch was cultivated without gas. Undoubtedly, C. sorokiniana MB-1 accumulated highest lutein content and lutein productivity when fed with CO2. Optimal sodium acetate concentration was 6.0 mg/L, resulting in maximal lutein content of 6.28 mg/g and lutein productivity of 3.06 mg/L/day. To further enhance lutein accumulation, semi-batch operation strategy was adopted. Determination of optimal medium replacement ratio was first conducted. The results show that 75% medium replacement ratio showed better efficiency in terms of lutein accumulation and was thus chosen for semi-batch cultivation. Using semi-batch with eight cycles, lutein productivity was much improved and reached up to 5.78 mg/L/d.
Lutein was then recovered by using both supercritical fluid extraction (SFE) and reduced pressure extraction method for comparison. Better results were obtained using reduced pressure extraction, while the highest recovery of lutein obtained from SFE was only 22%. Moreover, reduced pressure extraction can be applied at a lower temperature compared to conventional solvent extraction, thus preventing lutein degradation. In addition, solvent can also be recovered using rotary evaporator therefore minimizing the consumption of organic solvent. Recovery yield was found depending on the following parameters: pretreatment, pressure, solvent type, extraction time and temperature. Recovered lutein was measured using high-performance liquid chromatography (HPLC). High pressure cell disruption was chosen as pretreatment for being more effective than bead-beating. The results show that tetrahydrofuran (THF) is the most effective solvent for lutein recovery. With high pressure pretreatment, THF, and operation conditions of 850 mbar, 25°C and 20 min, about 87.0% lutein was recovered. Almost full lutein recovery was achieved at longer extraction time (40 min). However, since ethanol is more preferable in food processing, extraction and optimization using ethanol is also conducted. Using ethanol as solvent and under conditions of 450 mbar, 35°C and 40 min, 86.2% lutein was recovered.
The recovered lutein was then stabilized using vegetable oils. Shelf-stability of lutein in olive oil and sunflower oil was studied. Lutein suspended in oils were stored at 4 and 25°C. Samples were analyzed at selected times and kinetic data were used to estimate lutein stability and half-life. Samples were stored in dark and results showed that half-lives of lutein in olive oil at 4 and 25°C were 59.1 and 22.4 days, respectively. The corresponding results using sunflower oil at 4 and 25°C were 63.8 and 31.3 days, respectively. The reason behind the difference in stability could be the composition of antioxidant in the two type of oils. Vegetable oils were proven capable of prolonging shelf-life of lutein, compared to half-lives of lutein extracts in ethanol, which only lasted for 16.7 and 11.1 days, at 4 and 25°C, respectively.
論文目次 Acknowledgements VII
Contents IX
List of tables XIII
List of figures XIV
List of symbols XVII
Chapter 1 Introduction 1
1.1 Background 1
1.2 Motivation and purpose 2
Chapter 2 Literature review 4
2.1 Photosynthesis and photosynthetic pigments 4
2.1.1 Photosynthesis mechanism 4
2.1.2 Chlorophyll 6
2.1.3 Carotenoids 7
2.1.4 Lutein 9
2.2 Microalgae as lutein source 10
2.2.1 Microalgae 10
2.2.2 Benefits of using microalgae to produce lutein 12
2.2.3 Lutein biosynthesis 13
2.2.4 Function of lutein in microalgae 16
2.3 Factors affecting lutein productivity from microalgae 17
2.3.1 Carbon 17
2.3.2 Nitrogen 18
2.3.3 Medium composition 18
2.4 Lutein extraction from microalgae 21
2.4.1 Supercritical fluid extraction (SFE) 21
2.4.2 Reduced pressure extraction 23
2.5 Lutein stabilization and storage 25
Chapter 3 Materials and methods 27
3.1 Chemicals and materials 27
3.2 Equipment 28
3.3 Microalgae cultivation 29
3.3.1 Microalgae culture conditions 29
3.3.2 Operation strategy 30
3.4 Lutein extraction 31
3.4.1 Supercritical carbon dioxide (SCCO2) extraction 31
3.4.2 Reduced pressure extraction 32
3.5 Analytical methods 33
3.5.1 Determination of biomass concentration 33
3.5.2 Determination of residual nutrient concentration 34
3.5.3 Determination of biomass productivity 34
3.5.4 Determination of lutein content and lutein productivity 35
3.6 Stabilization of recovered lutein 37
Chapter 4 Results and discussions 39
4.1 Determination of optimal culture condition for the lutein accumulation of C. sorokiniana MB-1 39
4.1.1 Effect of different gases on lutein content and lutein productivity of C. sorokiniana MB-1 39
4.1.2 Effect of sodium acetate concentration on lutein content and lutein productivity of C. sorokiniana MB-1 41
4.1.3 Effect of different medium replacement ratio on lutein content and lutein productivity of C. sorokiniana MB-1 42
4.1.4 Effect of semi-batch operation on lutein content and lutein productivity of C. sorokiniana MB-1 44
4.1.5 Summary 47
4.2 Supercritical carbon dioxide extraction of lutein from microalgae C. sorokiniana MB-1 49
4.2.1 Effect of solvent on the extraction of lutein from microalgae C. sorokiniana MB-1 49
4.2.2 Effect of solvent ratio on the extraction of lutein from microalgae C. sorokiniana MB-1 50
4.2.3 Effect of temperature on the extraction of lutein from microalgae C. sorokiniana MB-1 51
4.2.4 Effect of pressure on the extraction of lutein from microalgae C. sorokiniana MB-1 52
4.2.5 Summary 53
4.3 Reduced pressure extraction of lutein from microalgae C. sorokiniana MB-1 54
4.3.1 Effect of pretreatment on the extraction of lutein from microalgae C. sorokiniana MB-1 54
4.3.2 Effect of solvent on the extraction of lutein from microalgae C. sorokiniana MB-1 56
4.3.3 Effect of extraction time on the extraction of lutein from microalgae C. sorokiniana MB-1 57
4.3.4 Effect of solvent reuse on the extraction of lutein from microalgae C. sorokiniana MB-1 58
4.3.5 Optimization of using ethanol as the solvent for the reduced pressure extraction of lutein from microalgae C. sorokiniana MB-1 59
4.3.6 Summary 61
4.4 Stability of recovered lutein suspended in vegetable oils 64
4.4.1 Stability of lutein in olive oil 65
4.4.2 Stability of lutein in sunflower oil 66
4.4.3 Summary 68
4.5 Vesicle encapsulation of lutein using microalgae-derived lipid 69
Chapter 5 Conclusion 72
References 75
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