New Perspective on the Interaction Behavior Between Riboflavin and β Lactoglobulin-β Casein Complex by Biophysical Techniques

Riboflavin (RF) is a vitamin that only exists in plants and microorganisms and must be procured externally by humans. On the other hand, there are two major allergic factors in cow's milk, including β-lactoglobulin (βLG) and β-casein (βCN), while their allergic properties can be eliminated by binding to micronutrients. In this regard, we examined the binding process of RF to βLG and βCN in the binary and ternary systems by different spectroscopies such as zeta potential, electric conductivity, and molecular modeling. According to the result of the fluorescence spectrum regarding the interaction of RF with βLG and βCN in binary and ternary systems, an increase in RF concentration declined the fluorescence intensity of three systems and also caused the quenching of proteins. Static quenching plays a pivotal role in the formation of stable interactions. The obtained thermodynamic parameters by Van't Hoff equation ascertained the predominance of hydrogen bonds and van der Waals interaction in all the systems. Considering how the negative value of ΔH⁰ resulted in the negative value of ΔG⁰, the systems were assumed to be enthalpy driven. The outcomes of circular dichroism (CD) disclosed that the attachment of RF to the targets of systems increased their a-helix content, which particularly included the binding of RF to βLG that led to the conversion of β-sheet to α-helix content. As indicated by the results of zeta potential, the low concentration of RF contained the dominance of hydrophobic forces in the interactions, whereas the enlargement of this concentration prevailed electrostatic forces. Moreover, conductometry measurements showed an extension in the rate of ionizable groups due to the addition of RF to the systems, which may increase the probability of an interaction between RF, βCN, and βLG in binary and ternary systems. In consistency with the outcomes of molecular dynamics simulation, the data of molecular docking approved the capability of RF in forming strong and stable interactions with βCN and βLG.