極大化利用大豆膠製備多烯磷脂膽鹼之研究

Abstract

多烯磷脂膽鹼(polyenylphosphatidylcholine, PPC)是一類具有生物活性的磷脂膽鹼(phosphatidylcholine, PC)，被認為具有保肝作用。本研究旨在探討最大化利用大豆膠以製備PPC。首先，本研究發展一種藉由RP-HPLC (reverse phase-high performance liquid chromatography)搭配ELSD (evaporative light scattering detector)定量卵磷脂中PPC分子組成的可行方法。分離條件係以C30管柱和乙腈/甲醇/三乙胺(40:58:2, v/v/v)等比移動相，流速1 mL/min，ELSD檢測之漂移管溫度為80°C和1.8 L/min的空氣流速。如同三酸甘油酯分子，我們發現 PC分子的理論碳數(theoretical carbon number, TCN)和滯留時間之間具有良好線性關係，可作為鑑定未知PC分子之依據。此外，由於PC分子的相對應答因子(relative response factor)彼此近似，因此，PC分子之含量可以其面積百分比代表。利用此分析方法分析大豆和葵花卵磷脂的PC分子組成，兩者均以1,2-dilinoleoyl-sn-glycero-3-phosphocholine (LLPC)、1-oleoyl-2-linoleoyl-sn-glycero-3-phosphocholine (OLPC)和1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC)為主，其中LLPC的含量分別為40.6%和64.3%。相較大豆來源，葵花來源的卵磷脂和油脂皆不含次亞麻油酸。第二階段，磷脂乙醇胺(phosphatidylethanolamine, PE)是大豆卵磷脂中含量僅次於PC的主要磷脂質，利用酵素催化轉磷脂醯基反應(transphosphatidy- lization)之手段可將PE轉換成PC。大豆膠經溶劑區分得到的大豆卵磷脂(95%酒精可溶物)，進一步利用矽膠管柱層析從大豆卵磷脂分離PE和PC，含量分別為86.4%和90.2%，回收率為71.8%和80.3%。Streptomycese sp.來源的磷脂酶D之磷脂醯基轉移能力較甘藍來源的磷脂酶D佳，可共價結合至戊二醛活化之磁性粒子成功製備固定化酵素。固定化磷脂酶D的最適反應pH值為7.0，反應溫度為37°C。探討基質濃度和鈣離子對固定化磷脂酶D催化轉磷脂醯基反應之影響，以確定最佳反應條件。轉磷脂醯基反應之最適條件係在含有120 mM鈣離子濃度，2.5 M膽鹼濃度和1活性單位(Unit, U)固定化磷脂酶D之乙酸乙酯/磷酸鹽緩衝溶液(pH 7.0)兩相系統，37°C下反應12小時，產物之PC轉換率可達到64.9%，同時有微量的磷脂酸(phosphatidic acid, PA)生成。固定化磷脂酶D在上述之最適條件下循環使用10次，仍無明顯的活性損失。然而，合成的PC其組成與大豆卵磷脂中的PC組成不同。主要以PLPC和LLPC為主，共佔72.4%，PPC含量為45.3%。第三階段，利用酵素性轉酯化反應(transesterification)合成結構PPC。反應材料的PC係利用矽膠管柱層析從大豆膠中回收與製備而得，製得之PC的含量為91.6%，回收率93.3%，LLPC含量為40.8%；另一反應物亞麻油酸係利用鹼水解葡萄籽油，並且搭配低溫溶劑結晶法製得，其產物之亞麻油酸回收率為25.0%，純度為87.5%。合成PPC之轉酯化反應所使用的固定化酵素為Lipozyme TLIM，探討最適化反應條件的結果顯示，反應溫度40°C、PC和亞麻油酸之莫耳比1:9和酵素使用量為20%的基質重量，不需額外添加水分，反應48小時後其PPC的含量為75.8%，產率為60.0%。此外，我們也將大豆PC以低溫溶劑區分方式，加入95%乙醇(1:25, w/v)在-7°C過濾，收集上清液發現PPC含量由40.8%提高至58.8%；沉澱部分再進行上述之酵素性轉酯化反應，將PPC含量從33.9%提高到72.2%。

Polyenylphosphatidylcholine (PPC), a subgroup of the bioactive agents in phosphatidylcholine (PC), has been indicated to possess liver-protective effects. This study aims to maximally utilize soybean gum to prepare PPC. First, we investigated a promising and feasible method to determine PC molecular species by reverse phase (RP)-high performance liquid chromatography (HPLC) equipped with an evaporative light scattering detector (ELSD). Chromatography was achieved using a C30 column and an isocratic mobile phase consisting of acetonitrile/methanol/triethylamine (40:58:2, v/v/v) at a flow rate of 1 mL/min, and ELSD detection was performed using 80°C for the drift tube and an air flow rate of 1.8 L/min. As triacylglycerol species, a linear correlation was observed between the retention time and theoretical carbon number (TCN) of individual PC species. In addition, owing to the relative response factors were close to each other, the content of PC species can be represented as the peak area ratio. The compositions of PC molecular species in soybean and sunflower lecithins were similar to each other, and the major PC molecular species were 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (LLPC), 1-oleoyl-2-linoleoyl-sn- glycero-3-phosphocholine (OLPC), and 1-palmitoyl-2-linoleoyl-sn-glycero-3- phosphocholine (PLPC). The contents of LLPC in soybean PC and sunflower PC were 40.6% and 64.3%, respectively. Second, phosphatidylethanolamine (PE) was one of the major phospholipids in soybean lecithin only lesser than PC. An enzymatic method was established to increase the PC portion by means of transphosphatidylization to convert PE to PC. PC and PE were separated from soybean lecithin (95% ethanol-soluble fraction) by silica gel chromatography with content of 86.4% and 90.2%, and recovery of 71.8% and 80.3%, respectively. Phospholipase D from Streptomycese sp. possessed better transphosphatidylization ability to convert PE to PC than phospholipase D from cabbage. Phospholipase D from Streptomycese sp. was immobilized by covalent binding to glutaraldehyde-activated magnetic particles. The optimum pH value and temperature of immobilized phospholipase D were pH 7.0 and 37°C, respectively. Reaction parameters of substrate and calcium ion concentrations were investigated to determine optimum conditions. The PC yield of the reaction product was 64.9%, achieved at 12 hours in the ethyl acetate/phosphate buffer (pH 7.0) system containing 120 mM Ca2+, 2.5 M choline chloride and 1 unit (U) of immobilized phospholipase D at 37°C. The immobilized phospholipase D could be repeatedly used for ten times and showed no significant activity loss. However, the PC molecular species compositions of synthesized PC were different from the origin PC in soybean lecithin. The synthesized PC was mainly composed of PLPC and LLPC, which were accounting for 72.4% while the PPC content was 45.3%. Last, structured lipid of PPC was synthesized from PC and linoleic acid. PC was obtained from soybean lecithin by silica gel chromatography with the content of 91.6% and recovery of 93.3%, moreover, the PPC content was 40.8%. Linoleic acid was prepared by low-temperature solvent crystallization with the purity of 87.5% and recovery of 25.0%. Lipozyme TLIM was selected to catalyze the acidolysis reaction. The optimal conditions were recommended to be 40°C, no water addition, substrate ratio 1:9 (linoleic acid/PC, mol/mol) and enzyme dosage 20% based on the total substrate weight. Under these conditions, the content of PPC was 75.8% and the yield of PC was up to 60.0%. In addition, the PPC content of supernatant could be increased from 40.8% to 58.8% after low-temperature solvent crystallization (95% ethanol, -7°C). The PPC content of residual PC fraction could be further elevated from 33.9% to 72.2% by acidolysis reaction.