基于聚集途径的热剪切诱导大豆蛋白纤维化机制研究

Texturized soybean protein is extruded with the meal after soybean defatted. The value of texturized soybean protein increases 20% from soybean kernel. Fibrous structure is an important quality characteristic and a price determination factor of extruded texturised protein. It indicates soybean protein forms denatured molecular aggregation during thermal shear processing, for differential scanning calorimetry enthalpy of soybean protein decreases and average molecular weight increases. There are two pathways of forming denatured molecular aggregation. Pathway 1 is subunits association or flocculation before molecular denaturation and then aggregation. Pathway 2 is molecular denaturation before coagulation or aggregation. The key question of this research is which main pathway and what type interaction mechanism are during the protein fibrosis induced by thermal and shear. Three dimensional macromolecular network of protein is stabilized and strengthened by electrostatic interactions, hydrogen bonding, hydrophobic interactions and disulfide bonds. Strengthening or breaking those interactions, soybean protein with different conformation, like subunits association or flocculation (presents pathway 1), complete unfolding(presents pathway 2), hydrogen-bonding-broken(presents mechanism 1), hydrophobic-interactions-broken (presents mechanism 2) and disulfide-bonds-reduction or disulfide-bonds-Oxidation (presents mechanism 3)partial denaturation, will be established. Analysis on association, denaturaion, aggregation of soybean protein with those kinds conformation, and comparing the fiber structure formation at the same time, the mechanism of fiber structure formation of soybean protein induced by thermal shear will be revealed. It is helpful for understanding subunits interaction, molecular unfolding and denatured molecular aggregation formation, and for controlling fiber structure formation of soybean protein during thermal shear processing.

大豆榨油后饼粕经过挤压生产组织化蛋白大约增值20%。纤维化程度决定着组织化蛋白的质量和价值。热转变焓值降低和平均分子量增加表明，热剪切诱导纤维化是蛋白质形成变性分子聚集体的过程。未变性亚基团聚后再变性，或分子去折叠变性后再聚集是形成蛋白质变性分子聚集体的两条途径。纤维化遵循哪条途径，以及通过哪种相互作用机制聚集是研究的科学问题。研究拟通过稳定或破坏维持蛋白质构象的作用方式，构建未变性团聚态（途径一）和完全变性态（途径二）蛋白质，模拟变性分子聚集体的形成，比较两条途径对纤维化程度的正负效应，确证大豆蛋白纤维化的途径。构建破坏氢键（机制一）、破坏疏水作用（机制二）、还原或氧化二硫键（机制三）等变性态蛋白质，检验其变性、聚集特性、热剪切诱导纤维化特性，比较其对纤维化程度的效应，分析氢键、疏水作用、二硫键对纤维化的贡献程度，揭示大豆蛋白热剪切诱导纤维化的机制。丰富和发展热剪切诱导蛋白质纤维化理论。

大豆榨油后饼粕经过挤压生产组织化蛋白，大约增值20%。纤维化程度决定着组织化蛋白的质量和价值。分子去折叠变性后再聚集可能是蛋白质纤维化的机理。项目设计了化学诱导剂和剪切共同诱导对大豆蛋白质聚集态的影响、热剪切诱导下蛋白质的聚集态变化、基于介观结构的剪切诱导蛋白质转扭稠度形成机理研究和突然停机法研究热剪切过程大豆蛋白质聚集态和纤维化机理。结果显示，亚硫酸钠诱导后，大豆蛋白二硫键断裂，游离巯基含量显著升高约2~5倍，聚集体解聚，相互缠结，表观粘稠度增大约1.4倍；而卵磷脂诱导后，会结合在大豆蛋白的疏水区域，表观黏度下降。而热剪切诱导后，十二烷基硫酸钠不溶性蛋白逐渐转变为可溶性蛋白，并且其含量与剪切单位机械能耗呈负相关，意味着疏水相互作用形成的蛋白质聚集体在减少。随着剪切时间的增加，蛋白质形状因子（回转半径Rg与水力学半径Rh的比值）从剪切前的~0.7–1.0 (<1min)，逐渐变为2.6（4.5 min）, ~2（9.0 min），表明蛋白质从球状逐渐转化为纺锤状。随后又降为1.1（35.0min）和1.4（55.0min），表明聚集体形貌又变为接近球状。多分散指数（重均摩尔质量Mw与数均摩尔质量Mn）的比值从剪切前的12.11，逐渐增加至18.1（9.0min），基本稳定在18.4（35.0min）和17.7（55.0min），表明蛋白质聚集体整体形貌分布的均匀性降低，异质性增加。蛋白质在高水分挤压纤维化过程中，从挤压机喂料端（一区）、机筒（二、三区）到模头（四、五区），蛋白质平均摩尔质量先升高后降低，但高于挤压前，表明蛋白质总体呈现聚集；挤压后二级结构中β-折叠含量明显升高，无规则卷曲含量明显降低，表明大豆蛋白经挤压后其分子结构变得更加有序；大豆蛋白的疏水相互作用被破坏，二硫键被破坏后又会重新生成。综上，大豆蛋白纤维化机理为蛋白质先解聚后聚集，最后聚集产生的应力松弛的过程，同时蛋白质二级结构和宏观上均变得更为有序。