Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
Biomacromolecules. 2009 Dec 14;10(12):3246-52. doi: 10.1021/bm900812x.
The formation of complexes between proteins and polysaccharides is of great importance for many food systems like foams, emulsions, acidified milk drinks, and so on. The complex formation between beta-lactoglobulin (beta-lg) and pectins with a well-defined physicochemical fine structure has been studied to elucidate the influence of overall charge and local charge density of pectin on the complex formation. Binding isotherms of beta-lg to pectin are constructed using fluorescence anisotropy, which is shown to be an excellent technique for this purpose, as it is fast and requires low sample volumes. From the binding isotherms the maximal adsorbed amount, binding constant (k(obs)) and the cooperativity of binding are obtained at different ionic strengths. The Hill model is used to fit the binding isotherms and is shown to be preferable over a Langmuir fit. At pH 4.25, k(obs) shows a maximum at an ionic strength of 10 mM when using a low methyl esterified pectin (LMP) due to the balance of attractive and repulsive electrostatic forces between beta-lg and pectin and beta-lg neighbors. For two high methyl esterified pectins, one with a blockwise distribution of methyl esters (HMP(B)) and one with a random distribution (HMP(R)), this ionic strength maximum is absent and k(obs) decreases with increasing ionic strength. k(obs) is found to be largest for LMP and HMP(B) and considerably lower for HMP(R). A positive cooperativity is observed for both LMP (above an ionic strength of 45 mM) and HMP(R) (above an ionic strength of 15 mM) but not for HMP(B). Positive cooperativity is thought to be caused by a rearrangement of the pectin helix structure caused by binding of beta-lg, thus creating new or binding sites with a higher affinity. To attain strong binding of beta-lg to pectin it is preferable to use a pectin with a blockwise distribution of methyl esters. When complex formation takes place in high ionic strength media an LMP gives the best results, while at low ionic strength a high methyl esterified pectin with blockwise distribution may give better results, due to reduced electrostatic repulsion between both pectin and beta-lg and beta-lg neighbors.
蛋白质和多糖之间复合物的形成对许多食品体系（如泡沫、乳液、酸化乳饮料等）非常重要。β-乳球蛋白（β-lg）与具有明确理化精细结构的果胶之间的复合物形成已被研究，以阐明果胶的总电荷和局部电荷密度对复合物形成的影响。使用荧光各向异性构建了β-lg 与果胶的结合等温线，结果表明该技术非常适合，因为它快速且需要的样品量低。从结合等温线中，可以在不同离子强度下获得最大吸附量、结合常数（k（obs））和结合的协同性。使用 Hill 模型拟合结合等温线，结果表明优于 Langmuir 拟合。在 pH 4.25 下，使用低甲酯化果胶（LMP）时，在 10 mM 的离子强度下 k（obs）达到最大值，这是由于β-lg 和果胶以及β-lg 相邻之间的吸引力和排斥力之间的静电平衡。对于两种高甲酯化果胶，一种具有块状甲酯分布（HMP（B）），另一种具有随机分布（HMP（R）），则不存在这种离子强度最大值，k（obs）随着离子强度的增加而降低。发现 LMP 和 HMP（B）的 k（obs）最大，而 HMP（R）的 k（obs）则大大降低。对于 LMP（离子强度高于 45 mM）和 HMP（R）（离子强度高于 15 mM）都观察到正协同性，但对于 HMP（B）则没有。正协同性被认为是由β-lg 结合引起的果胶螺旋结构的重排引起的，从而产生具有更高亲和力的新的或结合位点。为了使β-lg 与果胶具有强结合力，最好使用具有块状甲酯分布的果胶。当复合物在高离子强度介质中形成时，LMP 会产生最佳效果，而在低离子强度下，具有块状甲酯分布的高甲酯化果胶可能会产生更好的效果，因为果胶和β-lg 以及β-lg 相邻之间的静电排斥减少。
