富含EPA/DHA型结构磷脂的酶法合成条件优化及表征

为建立富含二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)型结构磷脂的制备方法,本试验以南极磷虾磷脂为原料,以EPA和DHA总结合率为指标,通过响应面法优化富含EPA/DHA型结构磷脂的酶法合成反应参数,并采用气相色谱(GC)和亲水作用色谱-质谱联用(HILIC-MS)技术对其结构进行表征。结果表明,最优酶催化反应条件为:底物质量比(游离脂肪酸:磷脂, m/m)5.15,反应温度55.22℃,水分添加量(以反应底物总质量计)0.92%,酶添加量(以反应底物总质量计)20%,正己烷3 mL,反应时间24 h,所得结构磷脂中EPA/DHA结合率达64.35%;经GC和HILIC-MS综合分析可知,结构磷脂中磷脂酰胆碱(PC)亚类40:10(20:5/20:5)(18.46%),42:11(20:5/22:6)(12.78%)和38:6(18:1/20:5&16:0/22:6) (7.07%)等分子种含量较高;溶血磷脂酰胆碱LPC亚类20:5(20:5/0:0)(34.40%),22:6(22:6/0:0)(25.99%)和O-16:0(O-16:0/0:0)(10.22%)等分子种含量较高;结构磷脂的PC和LPC组分中脂肪酸单链平均长度≥20的分子种相对含量较原料磷脂分别提高了33.12和37.28个百分点;结构磷脂的PC组分中双键数≥5和双键数≥10的分子种相对含量较原料磷脂分别提高了24.89和27.53个百分点,表明该方法有效提高了结构磷脂中EPA和DHA的含量。本研究结果为进一步探索和丰富酶促合成EPA/DHA型结构磷脂的机理和实践提供了一定的数据支持。

Enzymatic Preparation of EPA/DHA-Rich Phospholipids and Structure Characterization

In order to establish a synthetic method for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)-rich structured phospholipids , phospholipids of Antarctic krill was used as raw material, and the synthesis parameters of EPA/DHA rich structural phospholipids were optimized by response surface methodology. Gas chromatography (GC) and hydrophilic interaction chromatography and mass spectrometry (HILIC-MS) were used to analyze structures. The results showed that the optimal conditions were as follows: substrate mass ratio of free fatty acids to phospholipids 5.15, temperature 55.22℃, water addition 0.92 wt%, enzyme loading 20 wt%, hexane addition 3 mL and reaction time 24 h, under which the incorporation rate of EPA/DHA in the structural phospholipids was 64.35%. According to the results of GC and HILIC-MS, 40:10 (20:5/20:5) (18.46%), 42:11 (20:5/22:6) (12.78%) and 38:6 (18:1/20:5&16:0/22:6) (7.07%) were the main components of phosphatidylcholine (PC), while 20:5 (20:5/0:0) (34.40%), 22:6 (22:6/0:0) (25.99%) and O-16:0 (O-16:0/0:0) (10.22%) were the main components of lyso-phosphatidylcholine (LPC). Structured phospholipids with long fatty acids chains (average single chain length of fatty acids≥20) increased by 33.12% and 37.28% in PC and LPC, respectively. Phospholipids with highly unsaturated fatty acyl chains (double bonds≥10) increased by 27.53% in PC. The results demonstrated that this method could increase the content of EPA and DHA in structured phospholipids. This study provided theoretical foundation for further exploring and enriching the enzymatic synthesis mechanism and practice of EPA/DHA-rich structured phospholipids.