Glucose Glycation of α-Lactalbumin and β-Lactoglobulin in Glycerol Solutions.

The glucose glycation of α-lactalbumin and β-lactoglobulin at 50 °C in a glycerol-based liquid system was investigated to evaluate the effect of water activity on glycation and site-specificity in a glycerol matrix. Glycation extent during the reaction was determined using the o-phthalaldehyde (OPA) method as well as ultraperformance liquid chromatography combined with electro-spray ionization mass spectrum (UPLC-ESI-MS) analysis. Glycation sites were identified by data-independent acquisition LC-MS (LC-MSE). The surface potential achieved by PyMOL and the tertiary structure determined by circular dichroism (CD) were used to assist the analysis of the glycation site-specificity in the glycerol matrix. The water activity of glycerol solutions was negatively correlated to the glycerol concentration. Results showed that the initial glycation rate in glycerol matrix was fitted to a linear equation in the first 48 h. Glycation accelerated with the increase of glycerol concentration, namely, the decrease of water activity, regardless of the native structure of the protein. The glycation sites were identical at a similar DSP although achieved at different water activities, with 4 and 7 sites detected in α-lactalbumin and β-lactoglobulin, respectively. However, compared with the glycation sites in a water-based matrix, the site-specificity of glycation was affected by the glycerol matrix, depending on the native structure of the proteins. Glycation was prone to occur at the reactive sites distributed on the surface of the proteins, particularly in the region with positive potential.