EFFECTS OF LYOPHILIZATION ON THE OXIDATION OF WHEY PROTEINS

Lyophilization is among the most used drying methods for the preparation of stable proteins. However, despite the extensive use of lyophilization in several protein matrices, it can cause structural alterations in the proteins, mainly due to freezing step which could lead to oxidation. Oxidation can alter the physical-chemical and structural characteristics of the protein. Formation of carbonyl compounds and dityrosine can occur, consequently leading to polymerization and aggregation, altering the secondary and tertiary structures of the proteins and which will affect functional and nutritional properties. In addition, the consumption of foods with a high prevalence of oxidized proteins has been associated with the development of chronic-degenerative diseases, such as cancer. The main objective of this work was to investigate the effect of lyophilization on the oxidation of whey protein isolate (WPI). An amount of 20 mg/mL of WPI was dispersed in water by magnetic stirring for 30 min at room temperature and lyophilized (WPI-FD). WPI-FD and untreated WPI (WPI-C) were evaluated for turbidity, particle diameter, ζ-potential, SDS-PAGE electrophoresis, near infrared spectroscopy (NIR) and the levels of carbonyl groups, dityrosine and free SH groups were quantified. Protein carbonylation and dityrosine formation were the most altered in comparison to WPI-C - both markers were twice as many (P <0.05) in the WPI-FD. On the other hand, the content of free SH did not differ between the samples. Although the turbidity of WPI-FD was nearly 10 times lower than WPI-C, no differences were observed in the diameter of the particles and in the potential ζ (P >0.05), suggesting that structural changes during freezing/lyophilization may have contributed to aggregate formation. Under reducing conditions, SDS-PAGE showed no molecular weight changes or aggregate formation in WPI-FD. However, under non-reducing conditions, it has been observed high molecular weight aggregates, indicating that disulfide bonds are involved in the oxidation of WPI-FD. In the NIR spectra, it was observed difference between samples in the spectral region between 1040nm and 1110nm, corresponding to the third overtone of the NH and OH bond stretching, and between 1300nm and 1350nm, corresponding to the second overtone of the stretching of the CH bond. In the principal component analysis (PCA) obtained from the NIR spectra, the samples were completely separated, indicating that there were structural differences between the samples as a result of lyophilization. It is interesting to note that the structural changes observed by NIR reflected the results obtained for carbonylated protein and formation of dityrosine, suggesting that these oxidation reactions differentiated the samples. In conclusion, although the increase in carbonyl and dityrosine contents caused structural changes in the samples, such oxidative modifications were sufficient only to cause changes in turbidity.