Super resolution microscopy imaging of pH induced changes in the microstructure of casein micelles

Acidification of milk destabilizes casein micelles (CMs) and results in network formation. This process is fundamental during the manufacturing of dairy gels such as cheese and yoghurts. Understanding the structural alterations of CMs during this process aids to predict the physicochemical and sensory properties of dairy gels. Herein we utilize direct Stochastic Optical Reconstruction Microscopy (dSTORM) to visualize individual hydrated CMs and characterize pH-dependent changes in CM size distributions. CMs were immobilized, fixed, labelled with caseins-specific primary antibodies, and imaged by dSTORM using Alexa Fluor 647 conjugated secondary antibodies. While antibodies specific to κ- and β-casein were used to stain CMs, only β-casein antibodies enabled reproducible imaging of micelles across the entire chosen pH window. Furthermore, CM's structural evolution was studied at acidic pH values representing the conditions during acid milk gel formation and at three elevated pH values. dSTORM imaging of casein aggregates at pH 4.5, below the isoelectric point of caseins, showed that β-casein distribution throughout the protein network and resolved nano-sized pores within the structure. Moreover, automated and quantitative image analysis revealed that the average size of CMs increased upon alkalization to pH 7.5 and 8.3, whilst narrow size distributions were found upon acidification to pH 5.5.