Modification of plant and algal proteins through conjugation and complex coacervation for application as emulsifiers and encapsulants

Proteins are widely used as emulsifiers and encapsulants in food industry. In recent years, there has been a growing demand for non-animal proteins (particularly plant and algal ones) as alternative protein ingredients to be used as emulsifiers and encapsulants. This trend is driven by increasing consumer preference for sustainably sourced food ingredients and also due to increasing vegetarian and vegan lifestyle. However, the application of plant and algal proteins as emulsifiers and encapsulants is limited due to their relatively inferior solubility, emulsifying activity and interfacial properties. Covalent conjugation and/or non-covalent complexation (e.g., electrostatic charge driven complex coacervation) of protein with polysaccharide can be used to improve their techno-functional properties. For example, covalent bonding between the ɛ-amino group of lysine residues of proteins and the terminal reducing group of the carbonyl of polysaccharides produces conjugates, and these conjugates show improved solubility and emulsifying properties compared to unconjugated proteins. Complex coacervation occurs when positively charged proteins are brought in contact with negatively charged polysaccharides. Complex coacervates are also known to show better emulsifying properties compared to the uncomplexed proteins. Protein-polysaccharide conjugates and complex coacervates bring together the surface-active nature of proteins and steric hindrance and greater water solubility of polysaccharides and make them more effective as emulsifiers and encapsulants of unstable (yet valuable) compounds. There is a vast pool of knowledge on Maillard reaction driven covalent conjugation and complex coacervation of milk proteins and animal proteins (e.g., gelatin) with polysaccharides. There is a paucity of research on Maillard reaction driven conjugation of plant and algal proteins, especially when implemented through ‘wet-heating’ route. Wet-heating route involves carrying out Maillard reaction in aqueous medium under suitably chosen temperature and time and is known to be shorter and easy to control compared to conventional dry-heating route. There is no systematic research on complex coacervation of (already) conjugated proteins with polysaccharides. Furthermore, the efficacy of resulting complex coacervates of conjugated proteins as emulsifiers and encapsulants remains unknown. Therefore, this thesis aimed to gain greater insights into the Maillard reaction-induced conjugation of plant and algal proteins followed by complex coacervation. The practical significance of this work was to improve the emulsifying and encapsulating properties of plant and algal proteins by creating conjugates and their complex coacervates. Pea protein isolate (PPI) and Spirulina protein concentrate (SPC) were chosen as model plant and algal proteins. Maltodextrin (MD) and carrageenan (CG) were chosen as model polysaccharides for producing conjugates and complex coacervates, respectively. The Maillard-reaction induced conjugation of PPI and SPC with maltodextrin was optimised in terms of temperature and time so that the reaction remained confined to initial stage. The complex coacervation between SPC-MD conjugate with CG was optimised in terms of pH and protein-to-polysaccharide ratio. Both conjugates and complex coacervates were used as emulsifiers and encapsulants of oxygen-sensitive omega 3 rich oil and their effectiveness in encapsulating and delivering encapsulated oil to intestinal stage of digestion were investigated. The first experimental chapter was dedicated to implement and optimise the wet-heating route of Maillard reaction between PPI and maltodextrin (MD). The conjugation between PPI and MD, as well as the progression of Maillard reaction, were assessed by detecting the colour change and the formation of brown-pigmented melanoidins. The optimum reaction conditions in terms of pH, temperature and time were found to 8.0, 90 oC and 5 h, respectively. The Maillard reaction induced conjugation between PPI and MD was successfully controlled within the initial stage since there was no significant formation of melanoidins up to 5 h of reaction. The solubility, surface charge and emulsifying properties of the PPI were significantly improved after conjugating with MD. The second experimental chapter focused on the conjugation between Spirulina protein concentrate (SPC) and MD, based on the information obtained from the conjugation of PPI and MD. The glycation degree, molecular weight, secondary structure, solubility, surface hydrophobicity, antioxidant activity and emulsifying properties of the resulting SPC-MD conjugates were determined. The stability of oil-in-water (O/W) emulsions produced using SPC-MD conjugates as the emulsifier under thermal treatment was also evaluated. The conjugation of SPC with MD increased the solubility of SPC when the reaction was carried out for 6 h, beyond which the solubility decreased. The resulting SPC-MD conjugates significantly increased emulsifying properties as well as antioxidant activity. O/W emulsions stabilised by SPC-MD conjugates also showed higher thermal stability. The third experimental chapter investigated the complex coacervation between conjugated SPC (i.e., SPC-MD conjugates) and CG. The optimum conditions for the formation of complex coacervates were found to be (SPC-MD conjugate)-to-CG ratio of 24:1 (w/w) and pH of 3.0. The resulting (SPC-MD conjugate)-CG complex coacervates were then used as encapsulation wall materials to encapsulate canola oil at a core-to-wall ratio of 1:3 (w/w). The particle size, encapsulation efficiency, thermal stability, and oxidative stability of the resulting microcapsules were determined. The microcapsules produced using conjugate-based coacervates as encapsulants showed significantly higher encapsulation efficiency (92.5%) and lower surface oil content (1.52%) compared to those produced using complex coacervates of unconjugated SPC. The microcapsules produced using (SPC-MD conjugate)-CG coacervates also had much higher thermal and oxidative stability. The fourth experimental chapter focused on gaining insight into the in-vitro digestion behaviour of solid microcapsules stabilised by (SPC-MD conjugate)-CG coacervates. An in-vitro adult model that included oral, gastric, and intestinal stages was used for this purpose. The particle size, microstructure, free amino acid contents, molecular weight, released oil content and free fatty acid content at the end of each stage were determined. The conjugate-based coacervates, as encapsulating shell materials, were effective in protecting encapsulated oil against gastric digestion. Most of the encapsulated oil (62-67%) was released during the intestinal stage, primarily due to slow proteolysis of wall material. Lipolysis of released oil in the intestinal stage released up to 41.7% free fatty acids. Thus, (SPC-MD conjugate)-CG coacervates showed significant potential as wall materials to encapsulate oxygen-sensitive oils for their targeted delivery and controlled release in the intestinal stage of digestion. The findings from this thesis make a valuable contribution to the body of knowledge on the scientific aspects of Maillard reaction and complex coacervation, particularly in the complex coacervation of (already) conjugated protein.  The findings also establish that wet-heating route of Maillard reaction of plant and algal protein can be controlled within initial stage, and the formation of advanced glycation end products (AGEs) can be avoided. The conjugates and their complex coacervates of plant and algal proteins can be used as healthy and sustainable emulsifiers and encapsulants of oxygen-sensitive compounds, such as omega-3 rich oils, in the food industry.