Influence of pH and low/high- methoxy pectin complexation on the hydrophobic binding sites of β-lactoglobulin studied by a fluorescent probe method

Abstract

β-lactoglobulin(β-Lg)-polysaccharide soluble complexes formed in a specific pH range through electrostatic attraction have attracted a growing interest in the design of food-grade encapsulation systems for hydrophobic compounds, which is mainly ascribed to the ligand-binding properties of β-Lg. However, it remains unclear whether pH-induced conformational changes in β-Lg and its electrostatic complexation with anionic pectin affect their ability to bind hydrophobic compounds. Here, a fluorescent probe method was employed to provide useful insights into the field. Three solvatochromic fluorescent probes (i.e. Nile red, retinol and curcumin) were selected as representative models of hydrophobic compounds that were bound to the inner cavity or/and the outer surface of β-Lg. Binding with β-Lg or β-Lg/pectin complexes largely enhanced the fluorescence of the hydrophobic probes. Especially, the polarity difference of different binding sites on β-Lg was revealed by the fluorescence spectra of β-Lg-Nile red as a function of pH. Both Nile red and retinol were bound more favorably to β-Lg at neutral pH than at acidic pH, possibly due to the accessibility of the inner cavity in the former case. Upon acidification, the gradual reduction in fluorescence intensity of the pre-formed protein-ligands complexes (i.e. at pH 7.0) was ascribed to the dissociation between Nile red (or retinol) and the inner cavity of β-Lg. β-Lg-curcumin interactions were less affected by pH variations, suggesting curcumin mainly binding to the outer surface of β-Lg. For the three tested hydrophobic compounds, the formation of soluble complexes between β-Lg with pectin had no adverse effects on their interactions with the protein. These results may provide useful insights into the binding of hydrophobic compounds to β-Lg or β-Lg/pectin complexes. The methodology may also be extended to study the encapsulation performance of other biopolymers or particles.