酶催液中3'-唾液酸乳糖分离提纯方法研究

Study on the separation and purification method of 3’-sialyllactose in enzymatic solution

3’-唾液酸乳糖(3’-SL)是一种功能性母乳寡糖,在新生儿的免疫防御、炎症保护、肠道微生态构建中发挥重要作用。近年来,研究者通过构建特定酶系,实现3’-唾液酸乳糖的高产量酶催合成。以酶催反应液为原料,利用膜联用技术和脱色脱盐技术,对酶催液中的3’-唾液酸乳糖进行分离纯化,优化条件及结果如下:90℃热处理后过30nm膜超滤,压力250 kPa,滤洗2次;调定p H10.0后过3 000 Da膜超滤,压力600 kPa,滤洗5次;两步超滤下,蛋白截留率98.7%,目标物回收率87.4%。pH调至中性后过1 000 Da膜纳滤,压力1.9 MPa,滤洗7次,乳糖去除率达到90.7%。活性炭(ZX-305型)添加量1.2%,处理温度55℃,脱色率达到86.3%。离子交换树脂(001×7型和D315型)洗脱速率0.5 BV·h-1,料液3’-唾液酸乳糖浓度10 g·L^(-1),脱盐率达到94.1%,浓缩干燥后样品粉末纯度达到95%~97%(以干物质计)。样品通过液质联用仪、傅里叶红外光谱仪进行结构鉴定,确认为3’-唾液酸乳糖。

3’-sialylactose(3’-SL) is a functional breast milk oligosaccharide,which plays an important role in the immune defense,inflammatory protection,and intestinal microecological construction of neonates.In recent years,researchers have achieved high-yield enzymatic synthesis of 3’-SL by constructing specific enzyme systems.In this paper,the enzyme-catalyzed reaction solution was used as the raw material,and the 3’-SL in the enzyme-catalyzed solution was separated and purified by membrane technology and decolorization and desalination technology.The optimized conditions and results were as follows:heat treatment at 90 ℃,ultrafiltration through 30 nm membrane,at the pressure of 250 kPa,filtration washing twice;adjust pH to 10.0,and then filter through 3 000 Da membrane,at the pressure of 600 kPa,filtration washing 5 times;under two-step ultrafiltration,the protein retention rate was 98.7%,and the target recovery rate was 87.4%.Adjust pH to 10.0,filter through 1 000 Da membrane nanofiltration,at the pressure of 1.9 MPa,filtration washing 7 times,the lactose removal rate reached 90.7%.Under the conditions of addition amount of activated carbon(ZX-305) of 1.2%,and the treatment temperature of 55 ℃,the decolorization rate reached 86.3%.The elution rate of ion exchange resin(001×7 and D315)was 0.5 BV·h-1,the concentration of 3’-SL in the feed solution was 10 g·L^(-1),the desalination rate reached 94.1%,and the purity of the sample powder after concentrating and drying reached 95%-97%(on dry matter).The structure of the sample was identified by liquid chromatography-mass spectrometer and Fourier transform infrared spectrometer,and it was confirmed to be 3’-SL.