Complexation between flaxseed proteins, polyphenols and gums: mechanism and application
Creators: name: PHAM, Loc Bao
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300604
subject: Food packaging, preservation and processing

description: The interaction between proteins and polyphenols can produce complexes that can be used as emulsifiers and encapsulants for food application. Protein-polyphenol interaction can be of non covalent and covalent nature; the latter produces protein-phenolic conjugates/adducts with improved thermal stability, antioxidant activity and emulsion stability compared to the native protein. However, most of the reported studies on protein-phenolic interactions are performed using animal proteins. The studies conducted on plant protein-phenolic interaction such as to explain the astringency of wine, cloudiness in beer and certain fruit juices, which have negative implication, and cannot necessarily explain the positive outcome of protein-phenolic interaction in developing useful food ingredients. Given that the use of plant-based ingredients is becoming increasingly popular, it is of practical important to understand the covalent interaction between plant proteins and polyphenols, as well as the physicochemical and functional characteristics of the resulting conjugates for their potential use as novel food ingredients. Therefore, the main objective of this Thesis was to understand the mechanism of formation of plant protein phenolic adducts/conjugates, determine the optimum conditions under which these adducts produce complex coacervates with polysaccharide gum, and use the knowledge to produce emulsions and microcapsules of omega-3 rich oils. The resulting emulsions and microcapsules will have increased stability against oxidation, and improved controlled/targeted release of oil in simulated gastro-intestinal environment.<br><br> 

Protein, polysaccharide gum and polyphenol used in this study were extracted and purified from a single plant (flaxseed). Firstly, the covalent reaction between flaxseed protein isolate (FPI) with phenolic compounds (flaxseed polyphenol (FPP), small molecular weight phenolic compounds: ferulic acid (FA), and hydroxytyrosol (HT)] was investigated. The effect of conjugation of phenolic compounds with FPI on its physicochemical (molecular weight, conformational structure, and thermal stability) and functional (solubility, surface hydrophobicity, and antioxidant capacity) was studied. It was found that the degree and nature of conjugation depended on the structure of the phenolic compounds. HT was oxidised into hydroxytyrosol quinone and subsequently reacted with the nucleophiles in the side chain of FPI to form C-N and C-S linkages with its aromatic ring. The regenerated HT was re oxidised and reacted with a second side chain of FPI to form a cross-link. The dimerization of two HT quinones, each carrying one side chain of FPI, also produced a cross-link.  FA and FPP were oxidised to phenolate ions and subsequently formed semiquinone intermediate radicals which reacted with the amino or sulfhydryl side chain of FPI to form uncross-linked FPI-FA and FPI-FPP adducts. Overall, all FPI-phenolic adducts showed improved thermal stability and antioxidant capacity. The FPI-HT adduct appeared to have higher solubility in water than FPI-FPP and FPI-FA. These findings suggest that the plant protein-phenolic adducts can be used as plant-based emulsifiers.<br><br>

Secondly, the emulsifying and interfacial (diffusion, absorption and realignment) properties of cross-linked (FPI-HT) and uncross-linked (FPI-FPP) adducts were examined to explore their potential use as emulsifiers, compared with unmodified FPI. All the FPI-phenolic adducts exhibited similar surface activity to the unmodified FPI; however, the emulsion stability of emulsions stabilised by the adducts was weaker. Importantly, the emulsions stabilised by FPI-FPP and FPI-HT adducts had better stability against oxidation compared to that of the FPI-stabilised one. Given the substantially improved oxidative stability of FPI-FPP and FPP HT adducts, they can be considered as emulsifiers of polyunsaturated fatty acid (PUFA)-rich oils.<br><br>

Thirdly, flaxseed gum (FG) was used as the oppositely charged biopolymer to induce the complex coacervate with FPI-phenolic adducts. The optimum conditions for complex coacervation between these adduct, un-adducted FPI and FG were determined and found to be within a narrow pH range (4.6±0.1). The optimum protein-to-gum and adducts-to-gum ratio was also identical (6:1). Then the FPI/FG and (FPI-adducts)/FG complex coacervates were used as wall materials to encapsulate flaxseed oil (FO) at a wall:core ratio of 2:1 and the resulting microcapsules were spray dried into powder. Microcapsules produced using FPI/FG and (FPI adducts)/FG had similar irregular shape and wrinkled surface morphology. The (FPI-HT)/FG was found to be the most protective wall matrix to stabilise FO with the lowest surface oil (1%) and the highest microencapsulation efficiency (95.4%). The microcapsule produced using (FPI-FPP)/FG had the highest oxidative stability.<br><br>

Finally, the powder FO microcapsules were subjected to in-vitro digestion, and breakdown of the microcapsule and release of the FO in oral, gastric and intestinal stages was determined. These microcapsules exhibited significant resistance against digestion at the oral and gastric environments with a low degree of proteolysis and oil release; there was also insignificant change in particle size and microstructure. The microcapsules were substantially digested (break down of shell structure, release of FO and formation of free fatty acids (FFA) formed) intestinal stage. The (FPI-HT)/FG/FO microcapsule had the highest degree of oil release (80 %) and free fatty acid formation (FFA; 38.5%) the intestinal stage. The (FPI-FPP)/FG/FO capsule had the lowest extent of oil (66.3%) and FFA (28.9%) release. These findings suggest that the (FPI-FPP)/FG coacervate can be a promising delivery vehicle for PUFA-rich oils and other hydrophobic compounds to gastrointestinal system.
This thesis makes the following important contribution the body of knowledge: (1) Some phenolic compounds (e.g. HT) can crosslink plant protein molecules (e.g. FPI) while others (e.g.  FPP) can form covalent conjugate at their side chain but cannot produce crosslinks; (2) The interfacial and emulsifying properties of plant protein-phenolic adducts depends on the nature of the adducted phenolic compounds; (3) The stability against oxidation of emulsions stabilised by plant  protein-phenolic adducts can be substantially higher yet the such emulsions can be less stable compared to those produced using native (unconjugated) proteins; (4) The plant protein-phenolic adduct can be effectively used as wall materials for complex coacervate-based microencapsulation of unstable hydrophobic ingredients (e.g. omega-3 rich oils) as they impart promising controlled/targeted release properties.<br><br>