Laboratoire d'Oenologie et Chimie Appliquée, Université de Reims Champagne-Ardenne, URVVC-SE UPRES EA 2069, BP 1039, 51687 Reims Cedex 2, France.
Anal Bioanal Chem. 2011 Sep;401(5):1541-9. doi: 10.1007/s00216-011-4912-8. Epub 2011 Apr 5.
Chitinases and thaumatin-like proteins are important grape proteins as they have a great influence on wine quality. The quantification of these proteins in grape juices and wines, along with their purification, is therefore crucial to study their intrinsic characteristics and the exact role they play in wines. The main isoforms of these two proteins from Chardonnay grape juice were thus purified by liquid chromatography. Two fast protein liquid chromatography (FLPC) steps allowed the fractionation and purification of the juice proteins, using cation exchange and hydrophobic interaction media. A further high-performance liquid chromatography (HPLC) step was used to achieve higher purity levels. Fraction assessment was achieved by mass spectrometry. Fraction purity was determined by HPLC to detect the presence of protein contaminants, and by nuclear magnetic resonance (NMR) spectroscopy to detect the presence of organic contaminants. Once pure fractions of lyophilized chitinase and thaumatin-like protein were obtained, ultra-HPLC (UHPLC) and enzyme-linked immunosorbent assay (ELISA) calibration curves were constructed. The quantification of these proteins in different grape juice and wine samples was thus achieved for the first time with both techniques through comparison with the purified protein calibration curve. UHPLC and ELISA showed very consistent results (less than 16% deviation for both proteins) and either could be considered to provide an accurate and reliable quantification of proteins in the oenology field.
几丁质酶和覃蛋白是重要的葡萄蛋白，因为它们对葡萄酒的质量有很大的影响。因此，定量测定葡萄汁和葡萄酒中的这些蛋白质，并对其进行纯化，对于研究它们的固有特性以及它们在葡萄酒中的确切作用至关重要。本研究采用液相色谱法对霞多丽葡萄汁中的这两种蛋白质的主要同工型进行了纯化。两步快速蛋白液相色谱（FLPC），采用阳离子交换和疏水相互作用介质对果汁蛋白进行了分级和纯化。进一步的高效液相色谱（HPLC）步骤用于实现更高的纯度水平。通过质谱进行馏分评估。通过 HPLC 检测蛋白质杂质的存在来确定馏分纯度，并通过核磁共振（NMR）光谱检测有机杂质的存在来确定馏分纯度。一旦获得冻干几丁质酶和覃蛋白的纯馏分，就构建了超高效液相色谱（UHPLC）和酶联免疫吸附测定（ELISA）校准曲线。首次通过与纯化蛋白校准曲线的比较，利用这两种技术实现了不同葡萄汁和葡萄酒样品中这些蛋白质的定量。UHPLC 和 ELISA 显示出非常一致的结果（两种蛋白质的偏差均小于 16%），均可用于准确可靠地定量葡萄酒领域中的蛋白质。
