Elucidating Protein–Lipid Microstructure and Interactions in Hybrid Processed Cheeses Using Synchrotron Macro ATR-FTIR Microspectroscopy

Herein, synchrotron-based Fourier-transform infrared (S-FTIR) microspectroscopy was used to investigate the spatial distribution and molecular interactions within hybrid processed cheeses (HPCs) that contain casein and plant proteins. Unsupervised hierarchical cluster analysis (HCA) and k-means clustering provided consistent spatial differentiation, effectively separating the regions that were dominated by plant protein from those dominated by casein. Spectral analysis revealed protein–protein and protein–lipid interactions based on the type of plant protein used across plant-based cheeses and hybrid formulations. Cheese samples that contained only hemp protein isolate (HPI) were dominated by β-sheets (50%) and β-turns (21%), while mung bean protein isolate (MPI)-based cheeses exhibited considerable α-helix (35%) and β-sheet (48%) contents. In HPI–casein hybrid cheeses, hydrogen bonds predominated, with the amide II mode of proteins as the key discriminating component. By contrast, the amide I secondary protein structure was the primary marker that distinguished MPI from casein in MPI–casein cheeses. Increasing the casein content reduced plant protein aggregation and created a more heterogeneous environment, thereby supporting a flexible and disordered protein network. Furthermore, the S-FTIR map of lipids indicated that coconut oil was preferentially associated with casein rather than with plant proteins. Overall, these results highlight the role of S-FTIR microspectroscopy in resolving compositional and molecular-level interactions in hybrid cheese matrices. • Synchrotron-FTIR mapped spatial distribution of main components in hybrid cheeses. • HCA gave stable clusters and clear separation of casein and plant proteins. • Amide I/II bands identified plant proteins and casein in MPI- and HPI-casein cheeses. • Casein reduced plant protein aggregation and promoted the disorder protein network. • Synchrotron-FTIR and Raman spectroscopy provide complementary results.