This paper reports the potential of synchronous front-face fluorescence spectroscopy in the characterization at the molecular level of milk changes during mild heating from 4 to 50 °C and acidification in the pH range of 6.8 to 5.1. Synchronous fluorescence spectra were collected in the 250–550 nm excitation wavelength range using offsets of 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, and 240 nm between excitation and emission monochromators. The potential of parallel factor (PARAFAC) analysis in the decomposition of the whole synchronous fluorescence data set into the contribution of each of the fluorescent compounds present in milk has been investigated for heating and acidification data sets. Models were fitted from 1 to 7 components. Considering the core consistency values, PARAFAC models with three components have been considered. The first three components explained 94.43% and 94.13% of the total variance for heating and acidification data sets, respectively. The loading profiles of the first and second components derived from PARAFAC analysis performed on heating and acidification data sets corresponded quite well with the characteristics of tryptophan and vitamin A fluorescence spectra, respectively. The third component corresponded to the riboflavin fluorescence spectrum. Considering the heating experiment, the profile of the concentration mode for the second component showed large variations according to the temperature, which were assigned to the melting of triglycerides between 4 and 50 °C. For the acidification experiment, drastic changes in the concentration modes of the three components were observed for pH below 5.6, in agreement with structural changes in casein micelles.

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