灵芝多糖 (GLP) 因其多种药理作用而受到越来越多的关注。然而,只有在使用高度纯化和准确表征的 GLP 时,才有可能建立结构-生物活性关系。本研究开发了一种基于不对称流场-流分离 (AF4)、超滤和 Sevag 方法的组合技术,以改进 GLP 的纯化工艺。在整个研究过程中使用去离子水,避免了透析脱盐过程。此外,通过 AF4 耦合在线紫外-可见光 (UV)、多角度光散射 (MALS) 和差分示差折光 (dRI) 检测器 (AF4-UV-MALS-dRI) 对纯化 GLP 的分子量 (Mw) 和回转半径 (Rg) 进行了表征。结果表明,去除较大尺寸的蛋白质后,由于超滤和 AF4 分离机制的组合,在单次运行中获得了一个高度纯化的 GLP 组分 (多糖含量为 108.37 ± 0.45%) 及其结构信息(即 Mw、Rg 和表观密度)。结果表明,本研究开发的 GLP 分离、纯化和表征方法非常高效,有助于更好地了解其结构-功能关系。
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Asymmetrical flow field-flow fractionation combined with ultrafiltration: A novel and high-efficiency approach for separation, purification, and characterization of Ganoderma lucidum polysaccharides
Ganoderma lucidum polysaccharide (GLP) has attracted increasing attention owing to its multiple pharmacological effects. However, the establishment of a structure-bioactivity relationship is possible only if highly purified and accurately characterized GLP is used. In this study, a combined technique based on asymmetrical flow field-flow fractionation (AF4), ultrafiltration, and the Sevag method was developed to improve the purification process of GLP. Deionized water was used throughout this study which avoids the dialysis desalting process. Furthermore, the molecular weight (Mw) and radius of gyration (Rg) of the purified GLP were characterized by AF4 coupled online ultraviolet–visible (UV), multiangle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI). The results demonstrated that after removal of the larger size proteins, one highly purified GLP fraction (polysaccharide content of 108.37 ± 0.45%) and its structural information (i.e., Mw, Rg, and apparent density) were obtained in a single run owing to a combination of mechanisms of ultrafiltration and AF4 separation. The results suggested that the method developed in this study for separation, purification, and characterization of GLP is highly efficient, which could help to better understand its structure-function relationships.