Elucidation of molecular changes in milk induced by thermal processing using front-face fluorescence spectroscopy and FAST index analysis

Milk has been subjected to heat treatments to ensure quality and microbiological safety; however, heat treatments simultaneously induce molecular changes. The present study investigated the effects of different heat treatments on intrinsic milk fluorophores using front-face fluorescence spectroscopy, chemometric analysis, and the FAST (Fluorescence of Advanced Maillard products and Soluble Tryptophan) index. Raw milk samples were subjected to thermization (60 °C/15 s), low-temperature long-time (LTLT, 63 °C/30 min), high-temperature short-time (HTST, 72 °C/15 s), and boiling (100 °C/10 min). Fluorescence spectra of alkaline phosphatase, lactoperoxidase, β-lactoglobulin, tryptophan, and advanced Maillard products were recorded and analyzed to elucidate heat-induced molecular changes. Progressive increases in fluorescence intensity were observed for all fluorophores with increasing heat severity, reflecting protein unfolding, exposure of aromatic amino acid residues, and accumulation of fluorescent Maillard reaction products. The FAST index increased from raw milk (3.37) to boiled milk (15.34), demonstrating a strong correlation with cumulative thermal load and lysine losses. Principal component analysis explained 79.6% of the total spectral variance and discriminated milk samples according to heat treatment intensity, with distinct clustering of raw, thermized, pasteurized, and boiled samples. These results demonstrate that front-face fluorescence spectroscopy coupled with FAST index determination and chemometric analysis provides a rapid, non-destructive, and sensitive approach for assessing heat treatment intensity in milk, offering considerable potential for industrial process monitoring and quality assurance.