The effect of heat on the structure and aggregation behaviour of bovine B-lactoglobulins A, B and C : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University

Abstract

The bovine milk protein β-lactoglobulin (BLG) possesses a thiol group which becomes solvent exposed at elevated temperatures, leading to the formation of disulphide-linked milk protein aggregates. This phenomenon is of interest to the dairy industry because milk is heat-treated in many modern processes. This study is concerned with how the structure of BLG is altered during and as a consequence of heat treatment and how aggregates are formed. Bovine BLG exhibits genetic polymorphism and the A, B and C variants, present in New Zealand milks, differ in their susceptibilities to heat-induced structural change and aggregate formation, and their response to heat treatment is examined in the present study. This study used the following techniques: near and far UV CD, intrinsic protein fluorescence, hydrophobic probe fluorescence, thiol group solvent-exposure and both native-PAGE and SDS-PAGE. Spectroscopic and thiol exposure results suggest that the tertiary structure of BLG is altered during and as a consequence of heat treatment and that the amount of β-sheet in this protein does not alter appreciably as a consequence of heat treatment. PAGE results indicate that BLG forms a mixture of non-covalently-linked and disulphide-linked aggregates during heating, and that disulphide-linked dimers in particular are associated into larger non-covalently-linked aggregates. These non-covalently-linked aggregates are intermediates and large disulphide-linked aggregates are the end product of the BLG aggregation pathway. β-Lactoglobulin A forms aggregates, particularly large disulphide-linked aggregates, more slowly than BLGs B and C. Spectroscopic and thiol availability results suggest that the "intrinsic thermostability" of BLG C is appreciably greater than that of BLG B, which is slightly greater than that of BLG A. Furthermore, the extent of "irreversible structural change" in molecules of BLG C which occurs as a consequence of heat treatment is less than that in molecules of BLG A, which is less than that in molecules of BLG B. In the case of BLGs A and B this reflects the slower rate at which aggregates of BLG A form compared to that of BLG B. The present study has advanced the understanding of the BLG aggregation mechanism and how the A, B and C variants differ in their response to heat treatment.