Molecular Characterisation and Thermal Examination Of β-Lactoglobulin and 11S Glycinin with Hexanal – An Off-Flavour Compound
Creators: name: Ince, Cameron
Publisher: name: RMIT University
Year: 2025
Subjects: classificationCode: 300602
subject: Food chemistry and food sensory science

description: Despite the increasing interest to incorporate plant-based foods in western diets, recent sensory studies have revealed that taste and texture of plant-based protein source contain limitations when compared to current animal-based motifs. Methods such as UHT treatment have been implemented to reduce malodorous compound development but has proven to also impose bio-functional changes to the molecular structure of the overall protein molecule, an area which still requires extensive investigation.
In this PhD research, β-lactoglobulin (variant B) and 11S glycinin (extracted from raw soybean) were the chosen protein fractions to represent both animal-based and plant-based protein sources. The choice of ligand was analytical grade hexanal, a widely studied volatile aldehyde which is present in both protein systems. The surrounding pH for both systems was maintained at pH 7.2 due to their added molecular stability within these environments. Spectroscopical methods were chosen based on recent publications which have previously identified the binding characteristics of similar host protein fractions as well as the secondary structural examination upon protein and ligand interactions. A major theme throughout this PhD thesis is to compare the findings between animal-based and plant-based protein sources by performing uniform experimental procedures where appropriate which will be compared relative to one another throughout the following chapters.
The first two experimental chapters of this PhD thesis were performed in ambient conditions to set the foundation for the ensuing thermal treatment in the latter part of the work. A series of spectroscopical techniques were performed along with ensuing molecular docking techniques to probe the molecular transitions, secondary structural changes and binding characteristics between hexanal with β-lactoglobulin and 11S glycinin. A host of spectroscopical similarities were identified between the two host protein fractions which contained physical interactions stabilised by van der Waals, hydrogen bonding, pi-alkyl and alkyl interactions. However, incorporation of the complexed volatile compound resulted in slight observational differences between β-lactoglobulin and 11S glycinin. The initial signs were apparent with significant reductions and increases to differing secondary structural components between the two host protein fractions. Existing as a dimer, jobs plot analysis revealed a 2:3 binding ratio for the β-lactoglobulin fraction assuming three hexanal compounds, one at the interface located between the monomers of the β-lactoglobulin dimer and one in the calyx of each monomer. Existing as a trimer in this study, jobs plot revealed a 1:1 binding ratio between 11S glycinin and hexanal (2 per hexamer) with its top ranked biding pose located within the middle of chains A and B of the chain trimer in a ‘donut’ model. These findings suggest that despite uniform molecular transitions, and interaction type, binding characteristics may yet differ between the two host protein matrices which will be further investigated in the following chapters. 
Building from the first two chapters, chapters 3 and 4 contained a thermal procedure, to instigate potential covalent interactions between the host and ligand as seen in previous literature. The thermal stability of hexanal actuated the adopted thermal treatment parameters due to its volatility under elevated thermal conditions. Physical apparatus such as vacuum pressure tubes, glycerol bath and constant temperature logging were utilised to ensure the most optimal conditions for protein-ligand complexation. As such, elevated thermal conditions of 80˚C for an exposure time of 60 minutes was deemed the optimal conditions for both protein sources, to allow for complete protein-ligand complexation without precipitation. 
Performed experimental procedures followed the themes outlined in the first two chapters allowing a complete 4-way investigation of all experimental work provided in this thesis. MALDI-TOF/MS, UV-vis, CD and FTIR spectroscopic analysis were the chosen traditional benchtop analysis followed by GROMACS simulations to visualise such conditions using the crystal structures of β-lactoglobulin and 11S glycinin. Simulations depicted the thermally processed β-lactoglobulin crystal structure undergoes a conformational change, between 68 to 80˚C, a temperature that is within the Tanford transition range. For 11S, a similar area of interest was located upon concluding the simulated thermal procedure, where complete expansion of the β-sheet structures was evident within the individual chains of the 11S molecule. Molecular docking on the thermally simulated crystal structures revealed the top ranked binding positions have completely changed when examining the same crystal structures under ambient conditions as outlined in the first two experimental chapters. It is believed that within these new binding locales, an adjacent lysine amino acid residue has facilitated covalent interactions between the host and ligand. These findings are in large agreement with UV-vis and MALDI-TOF/MS spectra revealing a covalent reaction through a condensation reaction that has occurred between hexanal and the acidic subunit of 11S glycinin as well as all three protein variants of β-lactoglobulin. 
In summary, this PhD thesis deals with the binding interactions between the aliphatic aldehyde hexanal with the whey-based fraction β-lactoglobulin and soybean storage protein 11S glycinin. In ambient conditions, the binding mechanics reveal a moderate binding strength where only physical interactions occur between hexanal with β-lactoglobulin and 11S glycinin. However, alterations to the protein conformational state due to the imposed thermal treatment has resulted in new binding locations which were able to facilitate covalent interactions through a condensation reaction between hexanal and the new surrounding residues. Understanding these mechanisms between protein/aldehyde matrices is a significant step in developing a viable plant based commercial formulation to match the taste and odour of whey-based protein. Achieving this goal will improve consumer acceptance and enhance nutritional benefits which will lead to considerable marketing and business advantages.