Predicting hydrolysis of whey protein by mid-infrared spectroscopy

The possibility of using mid-infrared spectroscopy for prediction of hydrolysis of whey protein was explored. Two different protein concentrations (5% and 8%) were trypsinated at different enzyme-to-substrate ratios (1:100 and 1:200) and followed over time (0–8 h). Degree of hydrolysis was determined by use of a fluorescamine assay, whereas size exclusion chromatography (SEC) was used to quantify the loss of intact protein. The results show spectral changes as hydrolysis proceeded over time. Increased absorbance intensities in the region from 1588 to 1540 cm−1 as well as increased absorbance intensities around 1400 cm−1 were observed with increasing hydrolysis. These changes were assigned to formation of primary amines as well as an increase in carboxyl groups as hydrolysis proceeded. As the level of free amino terminals correlates with spectral changes, infrared spectroscopy is a promising tool for prediction of degree of hydrolysis of whey proteins.