Tcholakova Slavka, Denkov Nikolai D, Sidzhakova Doroteya, Campbell Bruce
Laboratory of Chemical Physics & Engineering, Faculty of Chemistry, Sofia University, 1164 Sofia, Bulgaria.
Langmuir. 2006 Jul 4;22(14):6042-52. doi: 10.1021/la0603626.
We present experimental results about the effects of thermal treatment, ionic strength, and pH on the protein adsorption and coalescence stability of freshly prepared (2 h after emulsification) and 6-day-stored emulsions, stabilized by the globular protein beta-lactoglobulin (BLG). In all emulsions studied, the volume fraction of the dispersed soybean oil is 30% and the mean drop diameter is d(32) approximately 40 microm. The protein concentration, C(BLG), is varied between 0.02 and 0.1 wt %, the electrolyte concentration, C(EL), between 1.5 mM and 1 M, and pH between 4.0 and 7.0. The emulsion heating is performed at 85 degrees C, which is above the denaturing temperature of BLG. The results show that, at C(BLG) > or = 0.04 wt %, C(EL) > or = 150 mM, and pH > or = 6.2, the heating leads to higher protein adsorption and to irreversible attachment of the adsorbed molecules, which results in enhanced steric repulsion between the protein adsorption multilayers and to higher emulsion stability. At low electrolyte concentration, C(EL) < or = 10 mM, the emulsion stability is determined by electrostatic interactions and is not affected significantly by the emulsion heating. The latter result is explained by electrostatic repulsion between the adsorbed protein molecules, which keeps them separated from each other and thus precludes the formation of disulfide covalent bonds in the protein adsorption layer. The coalescence stability of heated and nonheated emulsions is practically the same and does not depend on C(EL), when pH is around the isoelectric point (IEP) of the protein molecules. This is explained with the adsorption of uncharged BLG molecules, in compact conformation, which stores the reactive sulfhydryl groups hidden inside the molecule interior, thus preventing the formation of covalent intermolecular bonds upon heating. We studied also the effect of storage time on the stability of heated and nonheated emulsions. The stability of nonheated emulsions (C(BLG) = 0.1 wt %, C(EL) > or = 150 mM, and pH = 6.2) significantly decreases after 1 day of storage (aging effect). In contrast, no aging effect is observed after emulsion heating. FTIR spectra of heated and nonheated, fresh and aged emulsions suggest that the aging effect is caused by slow conformational changes of the protein molecules in the adsorption layer, accompanied with partial loss of the ordered secondary structure of the protein and with the formation of lateral noncovalent bonds (H-bonds and hydrophobic interactions) between the adsorbed molecules. After thermal treatment of the BLG emulsions, the molecules preserve their original secondary structure upon storage, which eliminates the aging effect.
我们展示了关于热处理、离子强度和pH值对新鲜制备的（乳化后2小时）和储存6天的乳液的蛋白质吸附及聚结稳定性影响的实验结果，这些乳液由球状蛋白β-乳球蛋白（BLG）稳定。在所研究的所有乳液中，分散的大豆油的体积分数为30%，平均液滴直径d(32)约为40微米。蛋白质浓度C(BLG)在0.02至0.1 wt%之间变化，电解质浓度C(EL)在1.5 mM至1 M之间变化，pH值在4.0至7.0之间变化。乳液在85℃下加热，该温度高于BLG的变性温度。结果表明，在C(BLG)≥0.04 wt%、C(EL)≥150 mM和pH≥6.2时，加热导致更高的蛋白质吸附以及吸附分子的不可逆附着，这导致蛋白质吸附多层之间的空间排斥增强以及乳液稳定性提高。在低电解质浓度下，C(EL)≤10 mM，乳液稳定性由静电相互作用决定，且不受乳液加热的显著影响。后一结果可通过吸附的蛋白质分子之间的静电排斥来解释，这种排斥使它们彼此分离，从而阻止了蛋白质吸附层中形成二硫共价键。当pH值在蛋白质分子的等电点（IEP）附近时，加热和未加热乳液的聚结稳定性实际上相同，且不依赖于C(EL)。这可以用紧密构象的不带电BLG分子的吸附来解释，这种分子将反应性巯基隐藏在分子内部，从而防止加热时形成分子间共价键。我们还研究了储存时间对加热和未加热乳液稳定性的影响。未加热乳液（C(BLG)=0.1 wt%、C(EL)≥150 mM和pH=6.2）储存1天后稳定性显著降低（老化效应）。相比之下，乳液加热后未观察到老化效应。加热和未加热、新鲜和老化乳液的FTIR光谱表明，老化效应是由吸附层中蛋白质分子的缓慢构象变化引起的，伴随着蛋白质有序二级结构的部分丧失以及吸附分子之间形成横向非共价键（氢键和疏水相互作用）。对BLG乳液进行热处理后，分子在储存时保持其原始二级结构，从而消除了老化效应。
