An iron binding assay to measure activity of known food sequestering agents: studies with buttermilk solids

A method enabling the measurement and differentiation of both ferrous (Fe2+) and ferric (Fe3+) binding was developed to assess the relative sequestering power of known iron chelators and buttermilk solids (BMS). Fe2+ and Fe3+ chelation by BMS (0.1–10.0 mg), phytic acid, ethylenediaminetetraacetic acid (EDTA) and citric acid (0.1 mM) was measured using a modification of a ferric-thiocyanate method. Fe2+ and Fe3+ was determined from the amount of unbound ferric-thiocyanate following incubation with chelator. The assay was optimized for polarity of solvent and pH. Fe2+ and Fe3+ binding by sequestering agents was, in descending order of affinity to be: phytic acid>EDTA>BMS>citric acid. Known food iron chelators had a higher affinity towards Fe3+, while BMS had a higher affinity towards Fe2+. BMS also demonstrated an excellent iron solubilization property through iron binding activity.