Probing the binding sites of bioactives with β-Lactoglobulin at different gastrointestinal pHs

Abstract

Encapsulation is an efficient strategy to preserve the nutritional value of bioactive compounds in functional foods. The whey protein, β-lactoglobulin (βLg), is a potential carrier for bioactives due to its stability over a range of pHs and in the presence of the gastric enzyme, pepsin. In this study, curcumin and folic acid are used as representative hydrophobic and hydrophilic bioactives compounds respectively to investigate the suitability of βLg to encapsulate at different pH conditions of the gastrointestinal tract (2.0, 4.0, and 7.4). Unexpectedly, we found through intrinsic fluorescence spectroscopy that βLg binds both curcumin and folic acid with similar binding affinity at all three pHs. Fourier-transform infrared experiments showed, that with an exception at pH 2.0, H-bonding play a role in the interaction of both bioactives with βLg. Circular dichroism spectroscopy and small angle X-ray scattering showed that the interactions does not alter the structure of the protein. Further, 2D-protein nuclear magnetic resonance spectroscopy demonstrated, that curcumin binds to an external groove at pH 2.65. Whereas at pH 4.0, curcumin was found to bind at the groove and contrary to expectations, also at the calyx (the EF loop was previously thought to close the entrance to the calyx at this pH). At pH 6.5, both curcumin and folic acid bind to βLg at the calyx and the groove. Overall, our study shows that βLg binds curcumin and folic acid at gastrointestinal pHs, suggesting its potential ability to encapsulate both hydrophobic and hydrophilic bioactive molecules in dairy functional foods.