pH and thermal processing effects on the conformational structure of β-Lactoglobulin in mixture with phenolic acids

<p>Microconstituents of dietary fibre (i.e., phenolic compounds) are known to possess strong antioxidant and radical scavenging activities, which contributes to the inhibition of many chronic diseases such as cancers, stroke, and coronary heart disease. On the other hand, dairy proteins are known as the main ingredients in liquid food formulations providing bio- and techno-functionality. ß-lactoglobulin is a major whey protein and its functionality and structure are highly dependent on the temperature and pH of the system. This protein can either exist predominantly as a monomer at low pH values or dimer under physiological conditions. Protein-phenolic compounds interactions in food and supplement systems have gained remarkable attention due to the widespread reports on the role of phenolic compounds in health and nutrition. However, it was found that the proposed health and nutritional effects of these compounds can be heavily affected based on their interactions with proteins in the same system. These interactions may lead to different effects including functionality alterations, precipitation and agglomeration of the proteins.</p> <p>However, previous literature contains insufficient data on the nature of molecular interactions between a protein, like ß-lactoglobulin, and phenolic compounds under acidic and neutral pHs or following heat treatment at high temperatures. Therefore, the main goal of this PhD study is to comprehensively investigate the nature of potential interactions occurring between ß-lactoglobulin and different phenolic compounds at both acidic and neutral environments as well as following heat treatment at an elevated temperature.</p> <p>In the first experimental chapter of this PhD, the binding between ß-lactoglobulin and ferulic acid were investigated at ambient temperature in relation to the monomer and dimer forms of the protein at pH 2.4 and 7.3, respectively. Furthermore, in order to take the inner-filter effect phenomena into consideration and eliminate this effect, a correction equation was used to correct for this effect prior to further processing the obtained fluorescence data. FTIR and CD analysis indicated that the secondary confirmation of ß-lactoglobulin was changed upon its complexation with ferulic acid, suggesting molecular interactions do happen in both the dimer and monomer forms. Furthermore, fluorescence quenching analysis and calculations indicated the presence of a binding site, with the dimeric complex producing weaker dissociation constant than the monomeric counterpart. Molecular dynamics simulations and docking studies showed that the preferred binding location of ferulic acid in the dimer form lay at the interface of the two monomers while the preferred binding site for the monomer was located within the calyx shaped ß-barrel structure of ß-lactoglobulin.</p> <p>In the second experimental chapter, research was advanced to investigate the effect of phenolic acid structure on the protein-phenolic compound interactions. These interactions were commonly investigated with inappropriate methodologies for spectroscopic analysis. The most common issues being overlooked are the inner filter effect and the use of unsuitable equations to calculate binding strength and stoichiometry, leading to the propagation of questionable methodology and reported results throughout the field. In this regard, molecular interactions between ß-lactoglobulin and vanillic acid were studied at ambient temperature and pH 2.4. Job plot and non-linear binding analyses were carried out on fluorescence data and it was found that the protein-phenolic acid binding stoichiometry for this system was 1:1 which was further supported by the obtained molecular modelling results. Furthermore, the contribution of water molecules to the stabilization of the complexes via the formation of protein-water-ligand, which were generally not accounted for in MD simulation analysis in previous works, were considered and assessed.</p> <p>The aforementioned findings led us to develop the next phase of this work and design the third experimental chapter to obtain further insight into the interactions between vanillic acid and the dimeric form of ß-lactoglobulin.In this regard, CD and FTIR measurements revealed remarkable changes in the secondary structure of ß-lactoglobulin upon its interaction with the ligand. Furthermore, fluorescence analysis showed that the dimeric complex with the phenolic compound produced a larger dissociation constant in comparison to what was observed for the monomeric counterpart at acidic pH. These observations confirmed that the phenolic compound structure could also have a noticeable effect on the binding site and strength of the ligand to the protein. Furthermore, job plot analysis identified the stoichiometry of 1:1 for the ß-lactoglobulin-vanillic acid complex at neutral pH which was in agreement with the obtained results of molecular modelling.</p> <p>The last experimental phase of this PhD explored the significance of heat treatment and high-temperature processing conditions on the type of molecular interactions between ß-lactoglobulin and phenolic acids. In this regard, the molecular nature of interactions between ß-lactoglobulin and ferulic acid was examined at pH 7.2 and following exposure of their mixture to high temperatures (121 oC). After that, the interactions between these two compounds were characterized using proteomic, multi-spectroscopic, and molecular modelling techniques. Unlike our observation in the previous experimental chapter, this time, UV-Vis showed a significant increase in the absorbance of ß-lactoglobulin following the heat treatment in the presence of the phenolic acid which suggested that ferulic acid could attach to the protein covalently. Moreover, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses confirmed the covalent interaction between the phenolic acid and ß-lactoglobulin after being subjected to heat treatment. Protein hydrolysis and MALDI-TOF measurements identified Lysine 100 as the binding site of ferulic acid to the heated protein. These observations were further supported by molecular docking studies and MD simulations where it was shown that a covalent bond was formed between Lys 100 residues of the protein and ferulic acid in order to create a new adduct. Moreover, the addition of the phenolic acid to the heated ß-lactoglobulin caused a considerable decrease in ¿-helix and ß-sheet elements of the protein, while increasing trends were observed for unordered chains and ß-turn components suggesting that the protein became less ordered upon this complexation at the elevated temperature. This work can provide a better understanding of the fate of different bioactive ingredients in heat-treated beverages and the effect of heat treatment on those products, allowing the industry to further improve their formulations and make informed decisions of the addition of insoluble fibres.</p>