MALDI-TOF-MS and in-depth dynamic simulations on the molecular forces determining the stability of the 4-hydroxybenzoic acid - ß-Casein complex following UHT-like treatment.

Previous work has suggested the ability of 4-hydroxybenzoic acid (4HBA) to bind to ß -casein following ultra high temperature-like processing (UHT) in model aqueous systems. The present work confirmed directly, using MALDI-TOF-MS, the presence of covalently bound 4HBA following UHT-like treatment. In subsequent molecular dynamics simulations, the 3D structure of the ß -casein molecule was modified so that the meta-C of 4HBA ring and the side chain amino group of lys32 were linked covalently. Such simulations further indicated that the covalent addition of the phenolic compound had impacted the protein density and solvent accessibility. Hydrogen bond analysis between lys32 and the remainder of the protein structure revealed that the covalent complexation supported the formation of additional hydrogen bonds. These increased from potentially 9, in the single protein molecule, to 51 in the complex with 4HBA. However, the persistence of hydrogen bonds was reduced, leading overall to decreased stability and increased protein flexibility.

Binding parameters and molecular dynamics of ß-lactoglobulin-vanillic acid complexation as a function of pH - part B: Neutral pH.

Interactions between the dimeric form of ß-lactoglobulin and vanillic acid were investigated at pH 7.2, using a variety of spectroscopic techniques and molecular dynamics (MD) simulations. FTIR and CD studies showed alterations in the secondary structure of the protein upon its interaction with the ligand. Fluorescence measurements indicated that the dimeric complex with the phenolic acid produced a large dissociation constant (KD) compared to the monomeric counterpart at acidic pH (part A of this series). Stoichiometry of 1:1 was identified for the ß-lactoglobulin-vanillic acid complex by Job plot analysis at neutral pH suggesting two ligand molecules can participate in binding with the dimer. Molecular docking and MD simulations suggested that the top-ranked binding sites of the ligand were located at the entrance of each ß-barrel structure of the dimer. These simulations also allowed identification of the contribution of water molecules, in the form of protein-water-ligand bridging interactions, to the complexes.

Binding parameters and molecular dynamics of ß-lactoglobulin-vanillic acid complexation as a function of pH - Part A: Acidic pH.

Protein-phenolic compound interactions are commonly investigated with inappropriate linear equations for the analysis of binding strength and stoichiometry. This work utilises more appropriate protocols for the investigation of molecular interactions between vanillic acid and ß-lactoglobulin at pH 2.4, where the protein predominately exists as a monomer. Non-linear binding and Job plot analysis were conducted on fluorescence data to effectively determine the interaction's dissociation constant (KD, 2.93 × 10-5 M) and stoichiometry (1:1). Furthermore, spectroscopic techniques revealed statistically significant alterations to the conformational characteristics of ß-lactoglobulin upon complexation. Molecular dynamics (MD) simulations support a 1:1 interaction stoichiometry and reveal that the stabilisation of vanillic acid was dynamic in nature but mainly supported by four π-alkyl interactions and one hydrogen bond, located within the ß-barrel of the monomer. Water molecules, which are generally not accounted for in MD simulation analysis, were shown to be an important factor in the ligand stabilization via bridging interactions.

Quantitative analysis of the phase volume of agarose-canola oil gels in comparison to blending law predictions using 3D imaging based on confocal laser scanning microscopy.

Studying the phase behaviour of composite gels facilitates understanding of their structural and textural properties at low and intermediate levels of solids. In this work, the phase behaviour of a model system of agarose including various concentrations of canola oil was studied. This was pursued using a variety of techniques including SEM, FTIR, microDSC and dynamic oscillation in-shear. The structural studies recorded strong, continuous agarose networks supporting soft, discontinuous canola oil inclusions, with increasing levels of canola oil strengthening the composite system. A novel confocal laser scanning microscopy (CLSM) method for quantitative in situ examination of the oil phase volume was developed using three-dimensional (3D) imaging and image analysis software - FIJI and Imaris. Microscopic observations were assessed in relation to theoretical predictions from rheology-based blending-law analysis. Quantitative outcomes from the combined 3D imaging and image analysis are in close agreement with the volume predictions for the oil phase obtained from the isostrain blending law indicating the suitability of this approach in quantifying the phase behaviour of composite materials. The results of this work indicate that the developed microscopic method shows promise and could be used in the determination of phase volume in more complex and industrially relevant systems.