高效凝胶色谱法同时测定普鲁兰多糖生物合成过程中分子量和含量

摘要

为了建立出芽短梗霉发酵过程中同时测定普鲁兰多糖分子量和含量的高效分子排阻色谱分析方法.采用保护柱(UltrahyfrogelTM6×40 mm)和凝胶柱(UltrahydrogelTMLinear 7.8×300 mm),流动相0.1 mol/L NaNO3,流速0.5 mL/min,柱温30℃,示差折光检测器(Waters 2424),以普鲁兰多糖分子量标准物进行分子量和含量标准曲线测定,利用建立的方法和标准曲线测定出芽短梗霉CGMCC No.11602发酵过程中普鲁兰多糖的分子量大小和含量.结果表明:普鲁兰多糖重均分子量(Molecular weight,Mw)在9.6 kDa~2350 kDa范围内,线性关系良好(R2=0.99),含量范围为0.2 g/L~1.0 g/L时,线性关系良好(R2=0.99),相比于传统的方法,利用发酵液同时测定普鲁兰多糖含量和分子量与传统测定方法结果无显著性差异(p>0.05).通过新的检测方法,不仅能够实现普鲁兰多糖发酵过程中含量与分子量的实时检测,也为可控性生产不同分子量普鲁兰多糖提供技术手段.%To establish a new and quick method for simultaneously determining of pullulan content and molecular weight by high gel permeation chromatography with the fermentation broth. A two-column set of Guard Column(UltrahyfrogelTM6 mm×40 mm)and gel column(UltrahydrogelTMLinear 7.8 mm×300 mm)was used at 30 ℃, with 0.1 mol/L NaNO3as the mobile phase at the flow rate of 0.5 mL/min. Refractive index detector 2 424 was applied.The molecular weight and content standard curve of pullulan were measured with molecular weight standard of pullulan. The molecular weight and content of pullulan, which was from fermentation liquid produced by A.pullulans CGMCC No.11602, were measured by establishing methods and standard curves.The average molecular weight(Mw)of pullulan ranged from 9.6 kDa-2 350 kDa(R2=0.99), and the content of pullulan ranged from 0.2 g/L-1.0 g/L(R2=0.99).Compared with the traditional methods,there were no significant differences for molecular weight and content by simultaneous determining of molecular weights and contents of pullulan during fermentation process with fermentation broth(p>0.05).This method not only solved the synchronous detection the content and molecular weight of pullulan during the fermentation process, but also accurately guided the different molecular weight of pullulan fermentation production in a large scale.