Thermal association of β-lactoglobulin AB (β-Lg) purified from Cheddar whey, a major source of commercial whey ingredients, was studied by dynamic light scattering (DLS). The objective was to observe the effect of the process and/or possible micromolecules in this β-Lg source of commercial relevance. Protein solution (8%, w/v) was heated (25−90 °C) at pH 3.5, 7, and 9.0. DLS data were analyzed according to the method of Cumulants for apparent mean diameter and by the Contin method for percentile size distribution. Data indicated gradual change in mean size that started at 35 °C, much below the reported denaturation temperature of 70 °C. Moreover, the percentile distribution of various micrometer- and submicrometer-sized aggregates differed at the different pH values studied, conceivably indicating conformational alteration limiting spatial freedom for intermolecular association. Monomeric/dimeric form (1−9 nm range) was seen only at pH 3.5 at temperatures below 65 °C. The ubiquitous aggregate size range was 100−599 nm (Agg3), and greatest changes in aggregate size and distribution were detected around 70 °C. Micrometer-sized (>1000 nm) aggregates were formed at this temperature and above, concomitant with disappearance of Agg3. On the basis of polydispersity data and rate kinetics dependent on the disappearance of Agg3, the association tendency between 25 and 70 °C was in the order pH 7.0 > pH 3.5 > pH 9.0. Keywords: Thermal; association; gelation; whey; β-lactoglobulin

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Freshly fractionated ␤-lactoglobulin AB (␤-Lg) from Cheddar whey was dispersed at pH 3.5, 7, and 9 buffers containing 20 mM EDTA, and circulated at 25˚C through a closed loop containing a 200 nm pore size membrane, to remove traces of dust and large aggregates, and an water jacketed cuvette placed within a Dynamic Light Scattering (DLS) device for real-time data acquisition. Filtered samples were step-wise heated from 25˚C to 90˚C with continuous data acquisition to study dynamic changes in mean aggregate diameter (MAD). Data were analyzed by cumulant method for apparent MAD and CONTIN for size distribution. Initial MAD (IMAD) of about 200 nm, reflecting the pore size of the filter used, was observed for all experiments prior to heating. Mid-range aggregate, Agg3 (100-599 nm), was ubiquitous in all distributions and Agg1 (monomer-dimer) was only seen at pH 7 and 25˚C. Increased temperatures gave rise to larger aggregates (>micron) (Agg4 and 5) with concomitant disappearance of Agg3. The greatest increase in MAD was at pH 3.5 and the lowest was at pH 9. Pre-gelation (<70˚C) kinetic rates were 4.29, 1.53, and -0.11¥ ¥ ¥ ¥10 -4 s -1 for pH 3.5, 7.0, and 9.0, respectively, giving MADs that were 3.5, 2.2 and 0.8 fold compared to IMAD. Above 70˚C (apparent gelation range), relative aggregate enlargement was greatest at pH 3.5 being 55, 21, and 4.2 fold of IMAD, respectively, for pH 3.5, 7, and 9 at 90˚C. An apparent gel formed at pH 3.5 and a turbid gel/coagulum formed at pH 7 and none at pH 9. It is conceivable that increased association at pre-gelation temperatures is required for gelation. A mechanism has been postulated.