Determination of apparent binding constants for aroma compounds with β-lactoglobulin by dynamic coupled column liquid chromatography

Apparent binding constants of aroma compounds limonene, alpha- and beta -ionone, and terpenyl acetate, with beta -lactoglobulin (BLG), were determined, using dynamic coupled column liquid chromatography, for pH values varying from 3 to 11. K-a values varied from 2.61 to 3.21 x 10(3) M-1 for limonene, indicating a strong interaction with BLG. Similarly, significant and close apparent binding constants were obtained for alpha- and beta -ionone, 1.7 x 10(2) and 4.5 to 5.4 x 10(2) M-1, respectively. These data indicated that a similar mechanism is involved for the binding of these two molecules. The weaker values obtained at low pH, for alpha -ionone relative to beta -ionone, can be explained by the existence of steric hindrance. An increase of the apparent binding constant was observed, for all the compounds studied, when the pH was increased from 3 to 9. At this pH, an apparent binding constant was obtained for terpenyl acetate (1.04 x 10(2) M-1), whereas this determination was not possible at pH 3 and 6. The apparent binding constant increase was in agreement with the decrease of aroma compound relative activity coefficient in the presence of BLG, previously observed at this pH. It indicated a best accessibility to the same binding site. The binding constants of all the aroma compounds studied decreased at pH 11 as a result of the important release of the BLG structure previously reported.