Improved Antioxidant Activity and Glycation of α-Lactalbumin after Ultrasonic Pretreatment Revealed by High-Resolution Mass Spectrometry

High-resolution mass spectrometry was performed to investigate the relationship between bovine α-lactalbumin (α-LA) subjected to ultrasonication and glycation treatment with respect to antioxidant activity. After α-LA was pretreated by ultrasonication combined with glycation, the treated α-LA showed low intrinsic fluorescence emission and high antioxidant activity at increased ultrasonic power levels. Prior to ultrasonic pretreatment, three glycated sites were identified, whereas the number of glycation sites was increased to four, four, five, and six after ultrasonic power at 60, 90, 120, and 150 W/cm², respectively, for 15 min. Thus, no obvious difference was found among the glycation sites at the ultrasonic power of 60 and 90 W/cm². The average degree of substitution per peptide molecule of α-LA was used to evaluate the glycation level per glycation site. All the samples pretreated by ultrasonication exhibited a higher glycation level compared with the untreated samples. Ultrasonic power at 150 W/cm² showed the most highly enhanced glycation extent and antioxidant activity. Therefore, the intensified glycation extent and the conformational changes of protein were responsible for the increase of antioxidant activity of α-LA. Moreover, high-resolution mass spectrometry is an efficient technique to understand the mechanism of the improved antioxidant activity.