Reply to the 'Comment on "Deciphering calcium-binding behaviors of casein phosphopeptides by experimental approaches...

The comment regarding a method we used to calculate the binding capacity of casein phosphopeptides (CPPs) with calcium. Dr. Horne stated that we overestimated the concentration of Ca²⁺. Indeed, peptides would bind with more calcium ions with the help of phosphate ions. However, in our statement, the binding capacity of CPPs with calcium we used included the binding with calcium in the presence of phosphate. In our solution, peptides sequester calcium phosphate to form soluble core-shell nanoclusters, preventing calcium phosphate precipitating. Even if the binding capacity of CPPs with calcium are overestimated as stated by Dr. Horne, these could not be a problem in deciphering calcium-binding behaviors. Phosphate was applied only in the optimizing and screening process presented in Fig. 1 and Table 1, other experiments (Fig. 2-5 in the commented article) did not include phosphate in the reaction system. Phosphate could not be a factor of these experiments.