Effects of Heat and High Hydrostatic Pressure Treatments on Disulfide Bonding Interchanges among the Proteins in Skim Milk

Traditionally, milk has been heat treated to control microorganisms and to alter its functionality, for example, to increase its heat stability. Pressure treatment has been considered as a possible alternative for microorganism control, but some of the functionality-related milk protein interactions have not been explored. The present study used two novel two-dimensional polyacrylamide gel electrophoresis (2D PAGE) methods to explore the differences in the irreversible disulfide bond changes among the milk proteins after four common heat treatments and after 30-min pressure treatments of milk at 200, 400, 600, and 800 MPa at ambient temperature (22 °C). The pasteurizing heat treatment (72 °C for 15 s) denatured and aggregated only a few minor whey proteins, but the high heat treatments (100 °C for 120 s, 120 °C for 120 s, and 140 °C for 5 s) formed disulfide-bonded aggregates that included a high proportion of all of the whey proteins and κ-casein (κ-CN) and a proportion of the αs2-CN. Pressure treatment of milk at 200 MPa caused β-lactoglobulin (β-LG) to form disulfide-bonded dimers and incorporated β-LG into aggregates, probably disulfide-bonded to κ-CN. The other whey proteins appeared to be less affected at 200 MPa for 30 min. In contrast, pressure treatment at 800 MPa incorporated β-LG and most of the minor whey proteins, as well as κ-CN and much of the αs2-CN, into aggregates. The accessibility of αs2-CN and formation of complexes involving αs2-CN, κ-CN, and whey proteins in the pressure treated milk is an important novel finding. However, only some of the α-lactalbumin was denatured or incorporated into the large aggregates. These and other results show that the differences between the stabilities of the proteins and the accessibilities of the disulfide bonds of the proteins at high temperature or pressure affect the formation pathways that give the differences among the resultant aggregates, the sizes of the aggregates, and the product functionalities. Keywords: High pressure; high temperature; protein aggregation; two-dimensional PAGE; disulfide bonds; β-lactoglobulin; κ-casein; αs2-casein; whey proteins; casein micelle