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Low-field nuclear magnetic resonance (NMR) spin-spin relaxation (T 2 ) measurements were used to study the denaturation and aggregation of β-lactoglobulin (β-LG) solutions of varying concentrations (1-80 g L −1 ) as they were heated at temperatures ranging from ambient up to 90• C. For concentrations of 1-10 g L −1 , the T 2 of β-LG solutions did not change, even after heating to 90• C. A decrease in T 2 was only observed when solutions having higher concentrations (20-80 g L −1 ) were heated. Circular dichroism (CD) spectroscopy and fluorescence tests using the dye 1-anilino-8-naphthalene sulfonate (ANS) on 0.2 and 1 g L −1 solutions, respectively, indicated there were changes in the protein's secondary and tertiary conformations when the β-LG solutions reached 70• C and above. In addition, dynamic light scattering (DLS) showed that protein aggregation occurred only at concentrations above 10 g L −1 and for heating at 70 • C and above. The hydrodynamic radius increased as T 2 decreased. When excess 2-mercaptoethanol was added, the changes in both T 2 and the hydrodynamic radius followed the same trend for all β-LG protein concentrations between 1 and 40 g L −1 . These observations led to the conclusion that the changes in T 2 were due to protein aggregation, not protein unfolding.

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