Separation and structural characterization of alpha-lactalbumin and beta-lactoglobulin from whey products

In most food applications, whey proteins are used, rather than the individual proteins and this accounts for the high functional variability among commercially available whey protein products, and limits their applications. The overall objective of this study was to investigate the structural and thermal properties of individual alpha-lactalbumin (alpha-Lac) and beta-lactoglobulin (beta-Lg) fractions isolated from different whey protein sources. A common non-chromatographic process that isolate alpha-Lac and beta-Lg, with relatively high purity and yield from liquid whey (LW), whey protein concentrate (WPC) and whey protein isolate (WPI) using different chelating agents, was developed. The use of sodium citrate (NaC) and sodium hexametaphosphate (SHMP) were more effective than other chelating agents. Yield results indicated that 47 to 69% of beta-Lg originally present in the whey preparations was recovered, with purities ranging from 84 to 95%, and protein contents ranging from 40 to 99%, while the yields of alpha-Lac were 23 to 89%, with purities ranging from 83 to 90%, and protein contents ranging from 65 to 96% depending on the source of whey protein preparations and type of chelating agents. Structural and thermal properties of beta-Lg and alpha-Lac isolated fractions were studied using polyacrylamide electrophoresis (native and SDS), RP-HPLC, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and electrospray ionization mass spectrometry (ESI-MS). Results showed that all beta-Lg and alpha-Lac isolated fractions exhibit increased thermal stability and reversibility over standard proteins and difference in thermal properties were dependent on protein source. The relative intensity of the 1692 cm-1 band in the beta-Lg isolated fractions was dependent on the nature of the chelating agent, and disappearance of this band occurred at temperature higher than that of beta-Lg standard, indicating increased thermal stability of beta-Lg isolated fractions. Denaturation of apo-alpha-Lac was related to the gradual decrease in the alpha-helix band and accompanied by the gain in intensity of 1653 and 1641 cm-1 bands, while denaturation of holo-alpha-Lac was associated by breakdown of beta-sheet structure and increase in turns and unordered structures. Changes in charge state distribution (CSD), as measured by ESI-MS of beta-Lg and alpha-Lac in response to pH and storage time, were only qualitative and were of relatively low resolution at basic pH. The hydrogen/deuterium (H/D) exchange results demonstrated that the conformation of holo-alpha-Lac was more stable than that of apo-alpha-Lac and conformation of beta-Lg variant B was more stable than beta-Lg variant A. Kinetics of H/D exchange indicated that alpha-Lac and beta-Lg fractions isolated from different whey protein sources have the same or improved conformational stabilities compared to that of alpha-Lac and beta-Lg standard. The covalent binding of 3 or more hexose residues to alpha-Lac enhanced its conformational stability, but covalent binding of two hexose residues to beta-Lg resulted in less stable conformation.