β-Lactoglobulin Nanofibrils and Their Interactions with Pectins

Abstract

A novel procedure which involves microwave heating and subsequent storage was developed to induce self-assembly of β-lactoglobulin into nanofibrils. Microwave heating at pH 2 markedly accelerated the self-assembly of β-lactoglobulin, resulting mature nanofibrils within 2 h. However, prolonged microwave heating led to the disintegration of nanofibrils. Storing the 2 h microwave heated β-lactoglobulin solution for 4 days led to increase the nanofibril yield to the maximum level that can be attained by conventional heating method. β-Lactoglobulin nanofibrils formed by both methods showed inter-strand and inter-sheet distances typical to amyloid fibrils. They also had a similar secondary structure component composition. Both nanofibrils were found to be typically made of 2 to 3 protofilaments, and these protofilaments had a similar diameter, irrespective of the method of nanofibril formation. However, the nanofibrils formed by the microwave method consisted of a higher proportion of large peptides, including intact β-lactoglobulin monomers, providing evidence for a different peptide composition from the conventionally made nanofibrils. The parts of peptides which are not involved in forming the nanofibril core were thought to exist as protrusions on the nanofibril surface. The nanofibrils formed by the microwave method, which contained large peptides thought to have more of these protrusions than the nanofibrils formed by the conventional method. β-Lactoglobulin nanofibrils formed by the both methods were then interacted with pectins having different degrees of methylesterification. Complex formation was observed due to the electrostatic interactions between nanofibrils and pectins at pH 2 and pH 3, and the morphology of the resulting complexes was dependent on the degree of methylesterfication of the pectin, pH and ionic strength of the medium. Among the different pectins interacted, a particular high methoxyl pectin was capable of laterally binding the nanofibrils into well-aligned ‘ribbons’. Specific enzymatic digestion of this pectin and analyses of the resulting products suggested that it contains a negatively charged, non-methylesterified galacturonic acid block in the middle of the molecule, with the possibility of having additional blocks near to the reducing and non-reducing ends of the molecule. Electrostatic interactions between these negatively charged blocks and β-lactoglobulin nanofibrils, either alone or together with hydrophobic interactions between the uncharged, methylesterified galacturonic acid chains of the pectin molecules are believed to cross-link the individual β-lactoglobulin nanofibrils into the form of ribbons.