Real-time measurement of heat stability of skim milk using attenuated total reflectance (ATR)-FTIR spectroscopy

Milk proteins are susceptible to denaturation and aggregation upon heating, affecting product quality and shelf-life. Understanding the underlying molecular changes during heating is important to the dairy industry for process optimisation and product functionality. This study used Attenuated Total Reflectance (ATR)-Fourier Transform Infrared (FTIR) spectroscopy to non-destructively measure changes in protein molecular structure as the precursor to heat-induced aggregation in milk. Raw skim milk was divided into three subsamples, adjusted to pH 6.2, native pH or pH 7. Each sample was heated at 85°C on a BioATR crystal, with scans taken at 1-min intervals over 20 min using FTIR to measure protein denaturation and aggregation. The second derivative of the amide I region was used to measure changes in protein structure, with the spectra for pH 6.2 samples changing faster than pH 6.8 or pH 7 samples, indicating a higher rate of denaturation. The peak at 1072 cm⁻¹ related to colloidal calcium phosphate (CCP) increased with increasing temperature and pH. More extensive changes in CCP between colloidal and serum phases and protein denaturation/aggregation correlated with lower heat stability in milk. This study highlights the potential of ATR-FTIR spectroscopy for assessing the heat stability of milk via in situ measurement of changes in protein structure and CCP.