- 學位論文
建立雨生紅球藻從培養到誘導其蝦紅素累積與萃取之流程
Building the Procedure from Culturing to Astaxanthin Induction and Extraction of Haematococcus Pluvialis
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摘要
雨生紅球藻是目前蝦紅素含量最高的微藻，為此我們建立了雨生紅球藻室內的培養流程並經由不同方法分析與萃取其蝦紅素萃取液以比較天然蝦紅素與合成蝦紅素之抗氧化能力。我們利用1-L PBR培養系統作為開始的優化條件找出最佳的培養液濃度與光強，之後利用20-L tank作為室內培養的放大最後結合兩者的綜合培養系統達到低起始濃度且高成長速率與高生物質產出。並且將含有蝦紅素的雨生紅球藻經由不同前處理後的蝦紅素萃取液與合成蝦紅素比較抗氧化能力。 第一階段為建立雨生紅球藻的培養優化，在1-L PBR首先以1x、1.5x與2x之不同濃度OHM培養基培養雨生紅球藻，再以30、60、90 µmol/m2/s之不同光強行篩選培養，最後得到1.5x OHM、 90 µmol/m2/s的條件下有最高的比生長速率與生物質產率分別為0.263 d-1與0.136 g/L/d，接續培養放大到20-L tank，利用20-L tank水流循環的優勢雨生紅球藻在過程中可以避免過多光照導致提早轉紅累積蝦紅素進入休眠，配合200 µmol/m2/s雙面的光照，以低到高不同的起始培養濃度OD682nm = 0.05、0.15與0.3 (0.08、0.16與0.24 g/L)培養後發現低起始濃度即可達到與其他二組同樣的生物質濃度，OD682nm = 0.05 (0.08 g/L)組別之比生長速率與生物質產率分別0.317 d-1與0.068 g/L/d，最後綜合20-L tank與1-L PBR的Combine system培養後的比生長速率與最終生物質濃度分別為0.309 d-1，與1.001 g/L，Combine system 能夠以較低得起始濃度達到1-L PBR的產量。在微藻達到生物質平緩期後以1-L PBR作為容器，300 µmol/m2/s 高光強誘導微藻轉紅。收集的藻粉以不同前處理以優化之後的萃取效率，結果以濃鹽酸處理後丙酮萃取的蝦紅素最多，其產量高達28 mg/g-Biomass。最後我們以DPPH、ABTS、FRAP與PFRAP抗氧化實驗評估不同蝦紅素來源包括實驗室萃取 (Asta_L)、市售健康產品(Asta_M)與合成蝦紅素(Asta_S)抗氧化結果，均得出抗氧化能力高到低分別為Asta_L、Asta_M與Asta_S。 這些結果指出天然酯類蝦紅素來源比起合成蝦紅素具有更高的抗氧化能力並且20-L tank與1-L PBR的Combine system是做為雨生紅球藻室內培養更為有效率的培養方式。
並列摘要
Haematococcus pluvialis is the microalgae species with the highest astaxanthin content. This experiment will establish an indoor culture process of Haematococcus pluvialis to the analysis of astaxanthin induction, extraction, and antioxidant capacity. We use 1-L PBR to establish the optimized culture condition, then enlarge the indoor culture with 20-L tank for a higher specific growth rate, last the 20-L tank and 1-L PBR were used as combined system. The astaxanthin-containing Haematococcus pluvialis cells were collected and extract after pre-treatment, the extracts were analyzed and the antioxidant capacity were compared with synthetic astaxanthin. The first stage was carried in 1-L PBR to optimized the culture condition for higher biomass productivity and growth rate. Different concentration of medium of 1x, 1.5x and 2x was compared, after that, we used different illumination of 30, 60 and 90 µmol/m2/s, the results shows culture condition under 1.5x OHM and 90 µmol/m2/s has the highest growth rate, biomass productivity of 0.263 d-1 and 0.136 g/L/d. Next, the culture was enlarged to 20-L tank, the circulation in the 20-L tank could prevent early cyst cell formation, with different initial biomass concentration under double-sided illumination of 200 µmol/m2/s, initial biomass concentration of 0.08、0.16 and 0.24 g/L were compared. Initial biomass concentration of 0.08 g/L biomass concentration has the best results, the growth rate and biomass productivity respectively were 0.317 d-1 and 0.068 g/L/d. The last we combine 20-L tank for its low initial biomass and high growth rate, and 1-L PBR for the high biomass in the stationary phase as a combined system. The growth rate and biomass concentration of the combined system respectively were 0.309 d-1, and 1.001 g/L. A combined system can produce the same biomass concentration as 1-L PBR with lower initial biomass. At the stationary phase, we use light intensity of 300 µmol/m2/s to introduce high-light induction at 1-L PBR. The freeze-dried biomass was treated with different pre-treatment, the results showed pre-treatment with HCl and extraction with acetone has the best astaxanthin concentration of 28 mg/g-biomass. At last, we examined the antioxidative power with DPPH, ABTS, FRAP and PFRAP assay to different astaxanthin of astaxanthin extracted from laboratory (Asta_L), market nutraceutical (Asta_M) and synthetic astaxanthin (Asta_S). The results of antioxidative power from high to low respectively were Asta_L, Asta_M, and Asta_S. These results indicated the nature astaxanthin as astaxanthin-esters is a superior antioxidant to synthetic astaxanthin and combined system is an efficient way for indoor cultivation of Haematococcus pluvialis.
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