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標題: 利用HPLC-ELSD對三酸甘油酯種類分析方法之建立
The analysis and method development of the triglyceride species by using HPLC-ELSD
作者: 辜鈺惠
Ku, Yu-Hui
關鍵字: edible oil;食用油;triacylglycerol;evaporation light scattering detector (ELSD);三酸甘油酯;光揮發散射偵測器
出版社: 食品暨應用生物科技學系所
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飲食攝取之油脂在人體生理上有許多功能,其脂肪酸多寡與組成之改變,會影響細胞膜及細胞核上的訊息傳遞路徑,進而去控制特定轉錄因子的活性表現。因此,飲食脂肪與許多慢性疾病及免疫反應失常有關。飲食中脂質約95%為三酸甘油酯(Triacylglycerol, TG),其它為磷脂質、膽固醇等。而三酸甘油酯之結構相當複雜,會因三酸甘油酯中三個脂肪酸的雙鍵位置cis/trans結構和R/S光學異構物而有不同結構。
本實驗利用HPLC-ELSD來分析食用大豆油之三酸甘油酯中三個脂肪酸種類,利用許多參數,包括:ECN、CN、DB、Log α及Log β,探討分析三酸甘油酯種類方法之可靠性,另外以批式發酵培養產油酵母菌Rhodotorula glutinis,探討發酵前活化菌體與發酵後菌體之三酸甘油酯中三個脂肪酸種類變化。
實驗結果顯示,分離不同食用油利用AlltimaTM HP C18 Hi-Load分析管柱有較好之解析度。在漂移管溫度40 ℃及霧化氣體流速之壓力20 psi 為ELSD偵測器最適條件。ELSD對油脂偵測極限可以達到1.25 μg,遠低於RI對油脂之偵測,修飾Goiffen et al.(1981)的方法,依照其參數應用在HPLC-ELSD對油脂分析,實驗結果顯示由於 ELSD不會有溶劑波峰出現,在理論推演時也不需加入內標,利用ECN、Log α、DB等參數,即可判定其三酸甘油酯種類。本實驗經由不同時間收集波峰收集液,再經由氣相層析分析確認比對之三酸甘油酯種類,其結果Intra-day小於3.50%,Inter-day小於2.26%,都可以使用HPLC-ELSD做油脂分析。
依上述方法測定R. glutinis活化菌體與發酵菌體之三酸甘油酯中三個脂肪酸的種類,發現發酵前與發酵後之三酸甘油酯種類不同,可能因微生物在限氮培養環境下,進而累積對生理有利之三酸甘油酯種類,其詳細原因還未了解,需進一步深入探討。

The dietary intake of fats in the body features many physiological functions. The amount of fatty acid and compositional changes will affect the physical characteristic of cell membranes and signal transduction path of the nucleus, and control the activity performance of specific transcription factors. The amounts of intake of dietary fats are related to a number of chronic diseases and abnormal immune responses. About 95% of the lipids in an average meal are constituted of triacylglycerol (TG), and the remainders are phospholipids, cholesterol and others. The structure of triacylglycerol is rather complex because the cis/trans double bond structure of the three fatty acids in TG molecules and the R/S optical isomerism come in different structures.
This experiment used HPLC-ELSD to analyze the three types of fatty acid contained in the triacylglycerol of edible soybean oil. We utilized several parameters including ECN, CN, DB, Log α and Log β to explore the reliability of the analyzing methods of triacylglycerol types, and on the other hand, also to cultivate oleaginous yeast (Rhodotorula glutinis) using batch fermentation method to explore the bacterial activation, before and after fermentation, on the changes of three fatty acid types in triacylglycerol.
The experimental results indicated that using the AlltimaTM HP C18 Hi-Load analysis column to separate different edible oils yielded a higher resolution. The optimal conditions for using ELSD are that of a drift tube temperature of 40 ℃ and nebulizer nitrogen pressure of 20 psi. The ELSD detection limit of fats reached 1.25 μg, which is well below the RI detection of fats and of which has a modified Goiffen et al.(1981) method. As the experimental results indicate, there is no occurrence of an ELSD solvent peak, so it is not necessary to add internal standard into the course of theoretical deduction, but only necessary to employ parameters such as ECN, Log α, and DB to identify its triacylglycerol types. During the experiment we gathered the peak collecting fluids at different time intervals using gas chromatography analysis to confirm the matching triacylglycerol types. Results show that the intra-day reading was lower than 3.5%, and that inter-day reading was lower than 2.26%, confirming that they are able to use HPLC-ELSD to analyze fats.
When the above-mentioned method was used to test the three fatty acid types of triacylglycerol during the process of R. glutinis bacterial activation and bacterial fermentation, we discovered that the types of triacylglycerol are different before and after fermentation. This might be due to further accumulation of triacylglycerol types towards physiological benefits when existing in the environment of nitrogen deficit cultivation of microorganisms. The detailed reasons for this have not yet been understood and still await in-depth exploration.
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