Impact of casein binding on thermal degradation of oxytetracycline: kinetics, products, and their toxicity

This work systematically investigated the effect of casein on the thermal transformation behavior of oxytetracycline (OTC). Fluorescence quenching and isothermal titration calorimetry experiments confirmed that OTC binds spontaneously to casein via non-covalent interactions. The -N(CH 3 ) 2 group in OTC was identified as the primary binding site. Casein-bound OTC was prepared using a combination of dialysis and ultrafiltration. During thermal treatment, the degradation of free OTC followed zero-order kinetics, whereas casein-bound OTC adhered to first-order kinetics. Seven identical thermal transformation products were identified, but the quantities of these products varied significantly depending on the existing form of OTC molecule. The binding of casein weakened the reactivity of the -N(CH 3 ) 2 group while enhancing the reactivity of degradation sites on the adjacent ring, resulting in decreased production of three lower-toxic products and increased production of four higher-toxicity products. Cytotoxicity assays revealed that heating increased the overall toxicity of OTC, particularly in its bound form.