Discriminating between different proteins in the microstructure of acidified milk gels by super-resolution microscopy

Super-resolution microscopy (Stimulated Emission Depletion, STED) combined with quantitative image analysis and rheology was used to study acidified milk gels with the addition of both endogenous milk proteins (i.e., liquid casein and whey protein concentrate, LCC and LWPC) and different types of whey protein ingredients. Casein and whey protein were stained separately using two different dyes prior to mixing. STED micrographs showed that added whey protein concentrate (WPC) and nano-particulated whey protein (NWP) were able to self-aggregate and attach to casein assemblies by inter-chain crosslinking. The behavior of NWP was shown to be similar to the behavior of LWPC, except that NWP formed larger aggregates with increased connectivity to the gel network. Micro-particulated whey protein (MWP) did not appear to interact with any other proteins and scattered MWP particles were visible in the mixed gels. The spatial colocalization of the fluorescence emission stemming from casein and whey protein, respectively, was highest for the system that contained only endogenous proteins. The lowest colocalization level was found for the systems with added MWP which also showed the highest image coarseness in accordance with the lowest observed G’. The systems with added LWPC or NWP exhibited thicker aggregate strands, which correlated to higher G’ compared to the other systems with WPC.