Amyloid protein aggregates of beta-lactoglobulin and their behavior along the process chain

淀粉样蛋白聚集体广泛存在于自然界中，以堆积的β-Sheet构象为特征。由乳清蛋白β-乳球蛋白(BLG)形成的淀粉样聚集体可以被特定地产生，以便利用它们作为一种功能物质的改变的物理化学性质。主要影响因素是高温、界面和/或高压引起的变性条件。同时，高应力(剪切、高压)和减小的Aw值(干燥)也会导致集料的碎裂。因此，在生物技术过程中，淀粉样蛋白聚集体可能会无意中生长，并导致不必要的沉积。另一方面，功能性淀粉样蛋白聚集体在加工过程中可能无法控制地降解，因此它们的尺寸分布和功能受到一定的不确定性。因此，为了过程控制，有必要了解淀粉样蛋白BLG聚集体的稳定性和聚集行为，以及生物技术过程中作用于淀粉样蛋白结构的建设性和破坏性作用力。基于BLG在均相溶液中的自缔合过程，用简化模型模拟了BLG从溶解到干燥的过程。将研究BLG的不同形态的淀粉样聚集体(纤维状、蠕虫状、球形)，以了解它们的组成、形成和稳定性。一个特别的重点是分析过程链中相关的物理和化学修饰的影响(例如，修饰的界面和剪切以及蛋白质氧化的影响)。单个氨基酸序列对自结合的意义是通过BLG的淀粉样蛋白产生片段和结构稳定的二硫键的靶向突变来实验理解的。实验结果将与数值模拟相结合，以获得力学方面的见解。另一个重点是研究不同淀粉样蛋白聚集体在干燥和成膜过程中的不稳定和稳定力。特别是，修改的界面(冰/空气)、pH变化和剩余盐的浓度被认为是相关的。因此，应分析各种干燥工艺对干燥和成膜的影响。干燥速度和稳定剂(低温保鲜剂、增塑剂)的不同是影响从中尺度(淀粉样聚集体)到宏观范围(薄膜中淀粉样聚集体的排列)的淀粉样结构的相关参数。

Amyloid aggregates occur ubiquitously in nature and are characterized by a stacked β-sheet conformation. Amyloid aggregates from whey protein β-lactoglobulin (BLG) can be specifically produced in order to take advantage of their altered physicochemical properties as a functional substance. The main influencing factors are denaturing conditions induced by high temperatures, interfaces and/or high pressure. At the same time, high stress (shearing, high pressure) and decreasing aW values (drying) also lead to fragmentation of the aggregate. Thus, amyloid aggregates in biotechnological processes can grow unintentionally and lead to unwanted deposits. On the other hand, functional amyloid aggregates can be degraded uncontrollably during processing, whereby their size profile and thus functionality is subject to a certain uncertainty. For process control it is therefore necessary to understand the stability and aggregation behaviour of amyloid BLG aggregates as well as the constructive and destructive forces acting on amyloid structures in biotechnological processes. Based on BLG's self-association process in a homogeneous solution, simplified models will be used to simulate the process from dissolving the BLG to drying. Different morphologies of amyloid aggregates from BLG (fibrils, worm-like, spherical) will be investigated to gain an understanding of their composition, formation and stability. A particular focus is the analysis of the effects of physical and chemical modifications that are relevant in the process chain (e.g. modified interfaces and shear as well as the influence of protein oxidation). The significance of individual amino acid sequences for self-association is to be understood experimentally through targeted mutations of amyloidogenic segments and structure-stabilizing disulphide bridges of BLG. Experimental results will be coupled with numerical simulation in order to gain mechanistical insights. Another focus is the investigation of destabilizing and stabilizing forces during drying and film formation of different amyloid aggregates. In particular, modified interfaces (ice/air), pH changes and concentration of remaining salts are considered relevant. Therefore, the influence on the drying and film formation by various drying processes shall be analyzed. Variances in drying speed and stabilizing additives (cryopreservatives, plasticizers) are relevant parameters that influence amyloid structures from the mesoscale (amyloid aggregate) to the macroscale range (alignment of the amyloid aggregates in the film).