肽类生物表面活性剂中消泡剂的分离研究

Research of the separation of defoamer in antimicrobial lipopeptides

本研究首次建立硅胶柱层析两步法以去除抗菌脂肽粗品中所含有的大豆油消泡剂成分,实验中采用高效液相色谱（HPLC）检测抗菌脂肽的含量,薄层色谱-可见光分光光度法定量分析消泡剂中甘油三酯含量,TLC法筛选大豆油的洗脱流动相。研究结果表明：使用200～300目的硅胶作为层析材料,硅胶的总负载量为12.5mg/g时,采取混合液（石油醚∶乙酸乙酯∶乙酸=20∶2∶1,体积比）为流动相,以1mL/min流速进行洗脱,可将97.97%的大豆油成分去除;第二阶段采用流动相为100%的乙醇以1mL/min流速进行洗脱可将抗菌脂肽解吸,抗菌脂肽的总回收率达到91.52%,纯度从34.07%提高到93.12%。本研究为肽类生物表面活性剂工业化生产和应用提供实验依据。

In this study,two- step method of silica gel chromatography was established to remove the defoamer in broth of antimicrobial lipopeptides for the first time.High- performance liquid chromatography（ HPLC） was applied to detect the concentration of surfactin. Thin layer chromatography（ TLC）- visible spectrophotometry was used to conduct quantitative analysis of triglyceride in eluent. Also,thin layer chromatography（ TLC） was taken as the way to select the developing agent for triglyceride. The result showed,when 200 ～300 mesh silica gel was used as the chromatography support beads,the total loading amount of silica gel was 12.5mg / g. Under the condition of the mixture（ petroleum ether∶ ethyl acetate∶ ethyl acid = 20 ∶ 2 ∶ 1,proportation of volume） as the fluid phase,flow rate of1 mL / min,97.97% of all the defoamer was obtained in the eluent. Secondly,when absolute ethyl alcohol was used as the fluid phase with a flow rate of 1mL / min,surfactin could be desorpted from the silica gel column,and its recovery reached 91.52%. The purification was greatly improved from 34.07% to 93.12%. This research provided experimental basement for the industrial production and application of antimicrobial lipopeptides.