臺灣博碩士論文加值系統

微藻中含有豐富的營養成分，其中除優質蛋白質外，也含有豐富的脂肪酸，尤其是水生物特有之 EPA (eicosapentaenoic acid) 及 DHA (docosahexaenoic acid) 具有營養與保健功效，故分析微藻脂肪酸組成具有營養上之重要性。然而，微藻的細胞壁厚需經細胞破壁才能完整的萃取脂肪，而超音波法是眾多細胞破碎中具快速、方便且安全的技術；此外，現今GC-MS (gas chromatography-mass spectrometry) 的應用性廣泛，且普遍存在於各分析場所，因此本研究將探討超音波細胞破壁輔助萃取方式用於改善傳統索氏與研磨珠法對微藻脂肪萃取步驟，並結合GC-MS 進行脂肪酸分析。研究首先建立以質譜作為偵測器之各脂肪酸相較於內部標準品之相對反應係數，各脂肪酸標準品與內部標準品在質譜中各濃度比值的線性回歸之相關係數皆大於0.99。比較微藻以索氏法萃取6個小時、乙醇-正己烷輔助研磨珠法以及超音波萃取20分鐘，得到脂肪酸量分別為4.99±1.77 mg/g、18.64±1.62 mg/g、20.01±1.82 mg/g，顯示索氏法萃取效果不佳，研磨珠法與超音波法具有相似的結果，但相較於超音波，研磨珠法需特殊設備且整體操作耗費2小時以上。本研究使用之超音波法在超音波處理後之樣品未分離脂肪，徑直將萃取液連同藻渣直接進行後續衍生化，結果顯示0、1、5、10、20、30分鐘超音波作用下，脂肪萃取含量並無差異，即使不處理 (0分鐘) 直接進行後續脂肪酸皂化與衍生化，也可以得到完整的脂肪酸含量，因此表明不經超音波，後續的皂化步驟應可以有效萃取脂肪，而為進一步的確認，遂將超音波萃取後之萃取液分離，得到脂肪含量僅4.11±2.43 mg/g，表示僅超音波並不能增加微藻脂肪萃取率。其後證明直接以脂肪酸分析皂化步驟，使用氫氧化鈉-甲醇溶液於80℃下加熱15分鐘，即可達到良好的脂肪萃取作用，並同時對脂肪酸進行皂化，遂據此方式開發出微藻中脂肪酸快速分析方法，該方法可以有效地從大家公認細胞壁厚且堅固的小球藻中萃取脂肪，並分析其脂肪酸組成。除此之外，透過分析數種微藻脂肪酸組成含量，並與文獻比較結果相符，顯示本方法具有良好的萃取與分析效果。綜合上述，微藻以本法處理再搭配GC-MS分析得以縮短整個分析時間，從傳統脂肪酸分析方法之6小時縮短至40分鐘。

Microalgae contain rich nutrients, including high-quality proteins and abundant fatty acids, particularly the aquatic-specific eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have nutritional and health benefits. Therefore, analyzing the fatty acid composition of microalgae is of nutritional importance. However, due to the thick cell walls of microalgae, cell disruption is necessary for complete lipid extraction. Ultrasonication is a fast, convenient, and safe technique for cell disruption among many methods available. Additionally, gas chromatography-mass spectrometry (GC-MS) is widely used and commonly found in various analytical settings. Hence, this study explores the use of ultrasonication-assisted extraction to improve the traditional Soxhlet and bead-beating methods for microalgae lipid extraction, combined with GC-MS for fatty acid analysis. The study first established the response factors of various fatty acids relative to an internal standard using mass spectrometry as the detector. The linear regression correlation coefficients of the concentration ratios of each fatty acid standard and the internal standard were all greater than 0.99. Comparing microalgae extracted using the Soxhlet method for 6 hours, ethanol-n-hexane-assisted bead-beating, and ultrasonication for 20 minutes, the fatty acid yields were 4.99±1.77 mg/g, 18.64±1.62 mg/g, and 20.01±1.82 mg/g, respectively, indicating that the Soxhlet extraction was less effective. Both the bead-beating and ultrasonication methods produced similar results, but bead-beating required specialized equipment and took over 2 hours in total. In this study, the ultrasonicated samples did not separate lipids prior to next step. The mixture was directly subjected to derivatization along with the algae residue. We found there is no difference in lipid extraction by ultrasonication between 0, 1, 5, 10, 20, and 30 minutes, even without ultrasonication (0 minutes). It was proposed that the subsequent saponification and derivatization steps by heated NaOH-Methanol solution may yield complete lipid extraction. For further confirmation, the extract after ultrasonication was separated, yielding a lipid content of only 4.11±2.43 mg/g, demonstrating that ultrasonication does not increase lipid extraction rates in microalgae. It was further proven that directly saponifying treatments of algae powder with a sodium hydroxide-methanol solution at 80℃ for 15 minutes effectively extracts lipids and saponifies fatty acids simultaneously. This method was thus developed into a rapid analysis technique for microalgae fatty acids analysis, capable of effectively extracting lipids from Chlorella, known for its thick and sturdy cell walls. Furthermore, this method was applied to analyze the fatty acid contents of several microalgae species, yielding results consistent with the literature, demonstrating good extraction and analysis performance. In summary, the analysis of fatty acids in microalgae by this developed rapid method can reduce the total analysis time from traditional 6 hours to 40 minutes.

摘要 I
ABSTRACT II
目錄 IV
圖目錄 VI
表目錄 VII
附錄 VIII
壹、前言 1
貳、文獻回顧 3
2.1 微藻 3
2.1.1介紹 3
2.1.2養殖現狀 3
2.1.3經濟價值 4
2.1.4影響微藻中脂肪酸組成之環境因子 5
2.1.4.1溫度 5
2.1.4.2營養源 5
2.1.4.3自營、異營和混合模式 6
2.2微藻中的脂肪酸 6
2.3微藻中脂肪之萃取法 8
2.3.1索氏萃取法 8
2.3.2 研磨珠破碎法 10
2.3.3 微波輔助脂肪萃取 10
2.3.4 高壓法 11
2.3.5超音波破碎輔助脂肪萃取 12
2.4脂肪酸分析 13
2.4.1 AOAC Official Method 996.06 13
2.4.2 AOCS Official Method Ce 1h-05 14
2.4.3 我國食品中脂肪酸之檢驗方法 14
參、研究材料與方法 16
3.1實驗架構 16
3.1.1 開發微藻中脂肪酸快速分析方法-不同萃取溶劑 16
3.1.2 開發微藻中脂肪酸快速分析方法-不同超音波破碎時間 17
3.1.5 脂肪酸於GC-MS之相對反應係數 (relative response factors, RF) 20
3.1.6 超音波加熱輔助破壁及脂肪酸衍生化操作步驟 21
3.1.7 微藻脂肪酸快速分析方法操作步驟 22
3.2研究材料 23
3.2.1微藻藻粉 23
3.3研究方法 23
3.3.1各脂肪酸甲基酯與內部標準品甲基酯於質譜偵測器之相對反應係數 23
3.3.2半自動索氏裝置萃取微藻中之脂肪酸 23
3.3.3研磨珠破碎微藻細胞壁並萃取脂肪之方法 24
3.3.4微藻經不同的萃取溶劑與超音波破碎時間之脂肪萃取方法 24
3.3.5微藻經不同超音波破碎時間後過濾藻粉取得脂肪萃取液之方法 25
3.3.6各種微藻之脂肪酸分析 25
3.3.7 脂肪酸氣相層析 25
3.3.8統計分析 26
肆、結果與討論 27
4.1各脂肪酸甲基酯與內部標準品甲基酯於質譜偵測器之脂肪酸相對反應係數 27
4.2半自動索氏萃取1-24小時對微藻脂肪萃取率之影響 36
4.3研磨珠破碎法對微藻中脂肪萃取之效用 39
4.4不同萃取溶劑對微藻中脂肪萃取之影響 41
4.5不同萃取技術對微藻中脂肪萃取之影響 44
4.6不同超音波破碎時間對微藻中脂肪萃取之影響 46
4.7微藻不經超音波萃取直接皂化後衍生化對其脂肪酸組成之影響 53
4.8數種微藻之脂肪酸分析 56
伍、結論 61
陸、參考文獻 62

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