Heat-induced interactions between hemp and milk proteins and their impact on emulsification : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Riddet Institute, Massey University, Palmerston North, New Zealand

Abstract

Hemp protein (HP) is a sustainable plant-based protein known for its high digestibility and favourable essential amino acid composition. However, like many plant proteins, HP suffers from low solubility, poor functional properties, and a high degree of aggregation in commercial powders, which limits its application in food systems. In response to the increasing demand for functional and sustainable food proteins, hybrid systems combining plant and milk proteins have emerged as promising solutions to overcome these challenges. This project focused on the development of functional hybrid protein ingredients by combining hemp protein (HP) with milk protein, particularly whey protein isolate (WPI), through a strategy involving heat-induced aggregation and microparticulation. The overall objective was to understand the mechanisms of heat‑induced interactions between HP and milk proteins, develop hybrid microparticles, and evaluate their emulsifying performance in both conventional oil‑in‑water emulsions and high internal phase emulsion (HIPEs) systems. Initially, dispersion of commercial HP was subjected to microfluidisation to improve dispersibility and reduce particle size. Heat treatment of microfluidised HP in the presence of β-lactoglobulin (β-lg) or whey protein isolate (WPI) resulted in the formation of irreversible disulphide-linked hybrid aggregates. Notably, WPI was found to suppress heat-induced aggregation of HP, highlighting its stabilising role during thermal processing. Comparative studies with sodium caseinate (NaCN) also suppressed heat-induced aggregation but showed that casein–HP interactions were reversible, likely driven by chaperone-like action, suggesting distinct binding mechanisms depending on the milk protein used. The influence of pH on heat-induced HP/WPI aggregation revealed that pH 8 promoted the formation of small, covalently stabilised aggregates, making it the optimal condition for hybrid microparticle formation. Well-dispersed HP/WPI microparticles were then developed via microparticulation, combining heat-induced aggregation at pH 8 and high-pressure homogenisation. Emulsions stabilised by microparticulated HP/WPI (≥1.5%, w/w) exhibited comparable emulsifying ability to WPI-dominated emulsions and significantly improved thermal stability. These hybrid microparticles were further tested in HIPEs systems, where they successfully stabilised emulsions. Compared to HIPEs stabilised by HP or WPI alone, those stabilised by microparticulated HP/WPI exhibited higher viscosity, greater storage modulus (Gʹ), and enhanced resistance to deformation, suggesting the formation of stronger internal droplet networks driven by protein–protein and droplet–droplet interactions. Importantly, the HIPEs stabilised by these hybrid microparticles also showed excellent heat stability, maintaining their structure, rheology, and appearance after heating. Overall, this project provided a new understanding of the heat-induced interactions between hemp and milk proteins and demonstrated a feasible approach for improving the functionality of hemp protein by forming hybrid protein systems with whey protein. Such findings will contribute to the development of novel functional food protein ingredients, enabling broader utilisation of plant proteins in emulsified and thermally processed food applications.