Plateforme TFFFC, Université de Toulouse, INP-PURPAN, Toulouse, France; Laboratoire de Chimie Agro-industrielle, LCA, Université de Toulouse, INRA, Toulouse, France.
SPO, INRAE, Univ Montpellier, Montpellier SupAgro, Montpellier, France.
J Chromatogr A. 2020 Oct 11;1629:461464. doi: 10.1016/j.chroma.2020.461464. Epub 2020 Aug 8.
Red wine is a complex matrix containing macromolecules such as condensed tannins and polysaccharides. Wine macromolecular components and their interactions have been reported to impact taste properties such as astringency but the colloidal systems formed in wine are not well known. A key prerequisite to characterize these systems is the ability to work under analytical conditions as close as possible to the colloid environment, preserving the sample structure and limiting the denaturation of macromolecular complexes. A method of Asymmetric Flow Field-Flow Fractionation (AF4) coupled with UV detection, multi-angle light scattering (MALS), and differential refractometer index (dRI) (AF4-UV-MALS-dRI) has been developed to analyse macromolecules, including tannins and polysaccharides, and macromolecular complexes, in red wine. This method separates objects according to their hydrodynamic radius and does not require calibration to determine molecular weight (M). AF4 can provide native separation of wine colloidal matter while working with simulated wine as mobile phase. The channel was equipped with a 350-µm spacer and the membrane made in regenerated cellulose had a cut-off of 5kDa. Different parameters of crossflow rate were investigated using a generic red wine to optimize separation conditions. Then, purified fractions of polysaccharides and tannins were analysed using the selected AF4 parameters. The comparison of the peaks obtained for these fractions and for the wine sample allowed us to determine the retention time associated with these macromolecules. The AF4 fractogram of wine was divided into four fractions. The first three were assigned to higher M tannins coeluted with lower M polysaccharides such as rhamnogalacturonan II (F1), to intermediate M polysaccharides (F2), and to higher M mannoproteins (F3) whereas the last fraction (F4) was not identified. Furthermore, our results have shown that AF4-UV-MALS-dRI could be an efficient technique to separate large size tannins as well as polysaccharides and macromolecular complexes.
红葡萄酒是一种含有大分子物质的复杂基质，如缩合单宁和多糖。葡萄酒的大分子成分及其相互作用已被报道会影响涩味等口感特性，但葡萄酒中的胶体系统并不为人所知。表征这些系统的一个关键前提是能够在尽可能接近胶体环境的分析条件下进行工作，保留样品结构并限制大分子复合物的变性。已经开发出一种不对称流场流分离（AF4）与紫外检测、多角度光散射（MALS）和差示折射计指数（dRI）相结合的方法（AF4-UV-MALS-dRI），用于分析红葡萄酒中的大分子物质，包括单宁和多糖，以及大分子复合物。该方法根据其流体力学半径分离物体，并且不需要校准来确定分子量（M）。AF4 可以在使用模拟酒作为流动相的情况下提供葡萄酒胶体物质的天然分离。通道配备了 350-µm 间隔器，再生纤维素制成的膜的截止值为 5kDa。使用通用红葡萄酒研究了不同的横流速率参数，以优化分离条件。然后，使用选定的 AF4 参数分析多糖和单宁的纯化馏分。这些馏分和葡萄酒样品的峰的比较允许我们确定与这些大分子相关的保留时间。葡萄酒的 AF4 馏分图被分为四个馏分。前三个被分配给与低分子量多糖（如鼠李半乳糖醛酸 II（F1））共洗脱的较高分子量单宁，分配给中间分子量多糖（F2）和较高分子量甘露蛋白（F3），而最后一个馏分（F4）未被鉴定。此外，我们的结果表明，AF4-UV-MALS-dRI 可能是一种有效的技术，可以分离大尺寸的单宁以及多糖和大分子复合物。
