Characterization of beta-lactoglobulin fibrillar assembly using atomic force microscopy, polyacrylamide gel electrophoresis, and in situ fourier transform infrared spectroscopy.

The aggregation process of beta-lactoglobulin (beta-lg) from 0 min to 20 h was studied using atomic force microscopy (AFM), scanning transmission electron microscopy (STEM), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and in situ attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Fibril assembly was monitored in real time using AFM up to 20 h. From 0 to 85 min, beta-lg monomers deformed and expanded with some aggregation. After 85 min, fibrillar structures were formed, exceeding 10 mum in length. Fibrillar structures were confirmed by STEM. Secondary structural changes occurring during fibril formation were monitored by ATR-FTIR at 80 degrees C and indicated a decrease in alpha-helix content and an increase in beta-sheet content. SDS-PAGE indicated that fibrils were composed of polypeptides and not intact monomers. In this study, beta-lg and whey protein isolate (WPI)-derived fibrils, including some double helices, in water were observed by AFM under ambient conditions and in their native aqueous environment.