Decoding co-encapsulation of β-carotene and tocopherol in zein nanoparticles through orthogonal spectroscopy and machine learning

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Protein-based nanoparticles (NPs), particularly zein NPs, are increasingly explored as oral carriers for nutrients and nutraceuticals due to their affordability, functionality, biodegradability, and biocompatibility. Co-encapsulation strategies can improve encapsulation efficiency and enhance the chemical stability of labile bioactive compounds; however, understanding the intermolecular interactions governing these systems remains challenging. In this study, zein NPs co-loaded with tocopherol (TC) and β-carotene (βC) at different ratios were investigated using Fourier Transform Infrared, fluorescence spectroscopy, and Raman hyperspectral imaging coupled with machine-learning analysis. TC incorporation enhanced both βC encapsulation efficiency and antioxidant activity. The 1:2 βC:TC ratio exhibited spectral signatures consistent with favorable intermolecular interactions, whereas a higher TC loading (1:3) showed spectral changes suggestive of perturbed non-covalent interactions. Raman spectral analysis further indicated that TC contributes to preserving βC stability under thermal conditions. These results advance understanding of co-encapsulation behavior and support the rational design of robust nanocarriers for labile nutraceutical compounds.