Biophysical insights into the encapsulation of Apo-lactoferrin for novel development of nutraceuticals

Increasingly, there has been consideration in the food and pharmaceutical industries on the development of novel active ingredients with multifaceted biological activities. This is due to rising global health awareness over emerging microorganisms that could cause serious ailments. Lactoferrin is a naturally occurring protein, isolated from the whey fraction of milk that exhibits diverse functionalities, including iron modulating capabilities as well as antibacterial properties. Among the three different types of lactoferrin, namely holo-lactoferrin, mono-lactoferrin, and apo-lactoferrin, the latter is more potent in suppressing the proliferation of iron dependent pathogens, including enterotoxigenic Escherichia coli and Listeria monocytogenes. This is primarily because of the iron sequestering capability of apo-lactoferrin. Interestingly, this additional advantage of apo-lactoferrin does not necessarily affect the growth of beneficial microorganisms, particularly from the genera of lactic acid bacteria as most of them can grow in the absence of iron. Thus, it is possible that a combination of apo-lactoferrin and lactic acid bacteria could offer a substantial improvement on the development of a novel nutraceutical. Nonetheless, the enhanced antimicrobial capacity of apo-lactoferrin also comes with a disadvantage as it has a lower stability compared to its other forms when processed at extreme temperatures during manufacturing or exposed to acidic conditions during gastric digestion. Therefore, essential measures are required in order to safeguard the stability of apo-lactoferrin, facilitating production at an industrial scale. In the food and pharmaceutical sectors, this is typically achieved by encapsulating apo-lactoferrin with another robust ingredient that could preserve its benefits. Whey protein isolate is one of the most promising encapsulating materials because of its nutritional value and broad physical as well as chemical attributes, including the capacity to undergo gelation and emulsification under different pH and temperatures. At present, studies have focused on various experimental procedures to stabilise lactoferrin through the formation of colloidal substances and encapsulation. However, the molecular mechanism underpinning these protection methodologies is not well understood. Accordingly, the aim of this research project is to implement state-of-the-art in silico methods to investigate the structural stability of apo-lactoferrin and its interactions with α-lactalbumin and β-lactoglobulin, two of the most copious components of whey protein isolate at simulated industrial processing conditions, including spray drying and freeze drying and at gastric pH. The objective of this project has also been extended into developing a fundamental model of a novel nutraceutical, composed of apo-lactoferrin and the peptidoglycan layer, representing lactic acid bacteria. This model could highlight the primary interactions that may play a role in the ability of apo-lactoferrin to serve as an encapsulating agent itself to protect lactic acid bacteria. In the first phase of the project, classical molecular dynamics simulations, free energy calculations, and network analysis were applied to evaluate and compare the structural stability and the binding of apo-lactoferrin to α-lactalbumin and β-lactoglobulin at spray drying (95 and 180 °C) and freeze drying (-20, 0, 30 °C) temperatures. The results exhibited that the whey protein components managed to protect the antibacterial peptide domains of apo-lactoferrin, lactoferricin and lactoferrampin. This is due to favourable electrostatic interactions between the negatively charged residues of α-lactalbumin and β-lactoglobulin and the positively charged residues of apo-lactoferrin that effectively hold apo-lactoferrin into a fixed conformation. Conversely, despite the stability of the anti-bacterial peptide motifs, apo-lactoferrin was found to be moderately denatured. This was shown in the secondary structure analysis that revealed partial transformation of helical elements into disorder coils when simulated at spray drying temperatures. In contrast, the original secondary structure of apo-lactoferrin when simulated at freeze drying temperatures remained largely intact, illustrating the sensitivity of apo-lactoferrin to heating. Therefore, simulations indicated that freeze dried apo-lactoferrin is more structurally stable than spray dried apo-lactoferrin. Nonetheless, network analysis depicted that the residues within apo-lactoferrin have the tendency to clump together when simulated under both temperature regimes. The reassociation of the residues, following exposure to these temperatures predicted that the protein would be easily dispersible in water during subsequent rehydration process. This is owing to the water molecules that may readily permeate into the protein’s interior to re-solvate clumped residues which were formerly separate. The findings of these studies demonstrated that processing apo-lactoferrin with α-lactalbumin and β-lactoglobulin as co-protectants under freeze drying condition may be more suited to preserving the function of apo-lactoferrin and produce more enhanced protein powders compared to that produced under spray drying condition. In the following phase of this project, the stability of the apo-lactoferrin whey protein oligomeric complex was simulated using advanced simulation techniques, encompassing atomistic molecular dynamics simulations, steered molecular dynamics simulations, and umbrella sampling calculations under a gastric pH of 1 to elucidate its possible unbinding mechanism and dissolution. It was discovered that the protein complex dissociates due to increased electrostatic repulsions as a result of the protonation of key residues under the acidic pH. Nevertheless, the free energy profile with respect to inter-protein distance determined the presence of multiple energy minima, demonstrating that these complexes, primarily composed of apo-lactoferrin and β-lactoglobulin units may favour the formation of flocculants and eventual gel creation. Practically, this property could be used to entrap additional bioactive compounds. Moreover, the antibacterial peptide segments in apo-lactoferrin are unaffected by the low pH, indicative of their potential viability after gastric digestion. In general, although the protein matrix was segregated under simulated gastric pH, intermittent contacts are conserved by apo-lactoferrin and β-lactoglobulin, which could be used as an encapsulant for further biological active ingredients. In the final chapter of this dissertation, an all-atom model, comprising apo-lactoferrin, α-lactalbumin, β-lactoglobulin, and a single disaccharide molecule, consisting of N-acetylglucosamine and N-acetylmuramic acid were constructed to approximate the interaction between the multimeric protein structure and the peptidoglycan scaffold of lactic acid bacteria. This model was simulated at spray drying temperatures of 68 and 110 °C, which are the optimised condition for producing spray dried Lactiplantibacillus plantarum, with whey protein isolate as the encapsulating agent. Simulations revealed that the peptidoglycan model interacts with the posterior N-lobe of apo-lactoferrin and the three-turn α-helix of β-lactoglobulin via van der Waals, electrostatic, and non-polar solvation interactions. Free energy calculations also ascertained that higher binding affinity is achieved at 110 °C compared to 68 °C, exhibiting that high temperatures are pivotal to generate strong interaction between the proteins and peptidoglycan structure via hydrophobic forces. Interestingly, simulations also highlighted that the secondary structure of apo-lactoferrin was maintained at these temperatures. Hence, it is plausible that processing apo-lactoferrin at a lower spray drying temperatures may produce apo-lactoferrin powders with enhanced efficacy compared that of at higher spray drying temperatures. In summary, computational modelling and simulations have been proven to be useful tools in obtaining insights at the molecular level, which enables in-depth understanding on the formation and dissociation of protein oligomeric systems as well as predictions of the assembly of novel biomolecules for engineering next generation nutraceuticals. Overall, the anti-bacterial properties of apo-lactoferrin were likely to be retained due to the protection conferred by the constituents of whey proteins under simulated spray drying, freeze drying, and gastric digestion. Nevertheless, as this dissertation has purely applied computational techniques to investigate the stability and encapsulation of apo-lactoferrin, future experimental studies are strongly recommended to corroborate the obtained findings. This includes further studies that explore the antibacterial capability of apo-lactoferrin as an enhanced nutraceutical agent in the food and pharmaceutical industries.