Quantifying microstructure and molecular dynamics in plant-dairy based model gels using FLIM and STED microscopy

The recent shift towards design of sustainable foods demands for developing advanced micro or more accurately, nanoscopic characterization techniques to study the complex structures formed in these foods. A well-studied and characterized model gel system, incorporating ingredients both from dairy and plant sources was prepared. Super-resolution STED microscopy and fluorescence lifetime imaging microscopy (FLIM) are combined to reveal the microstructure and molecular dynamics of acidified skim milk gels (ASMG) formulated with high-methoxy (HMP) and low-methoxy (LMP) pectins. A significant spatial variation of the fluorescence lifetime (τ) of the viscosity sensitive probe, Viscous Aqua (VA), is observed for all the samples in the sequence: τ(proteins) < τ(interface) < τ(voids). This suggests a more pronounced restriction of molecular dynamics in the void regions relative to the protein-dense regions. The lifetime distribution responds to the incorporation of pectin mainly in the protein regions of the gels. This indicates that pectin mainly localizes to the vicinity of the casein micelles. Spatial autocorrelation analysis of STED images provides novel insights into how pectin alters the ASMGs microstructure reveling distinct patterns of structural organization, with the correlation length ξ indicating the thickness of the protein network and the repeat distance λ representing the size of void regions within the gels. The incorporation of pectin leads to noticeable increases in both these parameters. The increase in ξ suggests a thicker, more cohesive protein network, likely due to pectin's interaction with casein micelles, expanding the protein-rich areas. Similarly, the rise in λ indicates larger void spaces between the protein regions, reflecting a more porous gel structure. The results highlight changes in the organization of water present inside the microstructure as induced by pectin incorporation into these gels and demonstrate the potential of integrating microstructure imaging and molecular dynamics to optimize similar plant and/or dairy based sustainable food systems.