Structure-function relationships in plant- and hybrid-protein processed cheese analogues : identifying drivers of functionality : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatū, New Zealand

Abstract

Processed cheese was developed by blending natural cheese with emulsifying salts and applying heat, resulting in a stable product with an extended shelf life and versatile functionality across a wide range of applications. However, growing concerns regarding environmental impact, sustainability, health, and animal welfare have driven increasing interest in plant-based alternatives. As a result, the development of plant-based processed cheese analogues has accelerated, aiming to deliver comparable functionality and sensory properties while addressing these broader societal demands. However, most commercial plant-based processed cheese analogues (PCAs) are primarily formulated with starch and vegetable oils. While starch contributes to viscosity and forms a gel-like network upon gelatinisation that can partially mimic dairy cheese structure, these systems often have low nutritional value and exhibit limited texture and functionality. In addition, substituting animal proteins with plant proteins remains challenging due to fundamental structural differences and functional limitations. Although the incorporation of plant proteins has led to some improvements, current formulation strategies remain largely empirical, and the underlying structure–function relationships are not well understood. Furthermore, the development of hybrid systems combining plant and milk proteins remains limited, and the mechanisms governing structure and functionality in such multiprotein systems are poorly defined. To address these limitations, this doctoral thesis investigated the incorporation of plant proteins into processed cheese model systems, with a focus on their role in structure formation and functionality. Specifically, it aims to elucidate structure–function relationships in plant-based and hybrid cheese analogues at the multiscale and to develop strategies for modifying plant proteins to modulate their interactions and aggregation behaviour, thereby tailoring network formation and improving functional performance, particularly in terms of microstructure, texture, meltability, and stretchability. First, various plant proteins were evaluated within plant-based processed cheese analogues to elucidate structure–function relationships. The results demonstrated that plant-based processed cheese analogues generally lacked a continuous protein network, leading to poor melting and stretchability. Microstructure and textural properties were strongly dependent on protein type and aggregation behaviour. Based on their distinct functional performance, mung bean protein and hemp protein were selected for further investigation in hybrid processed cheese systems formulated by blending with rennet casein at different ratios. The microstructure, texture, melting behaviour, and stretchability of these hybrid systems were characterised. The result indicated that both the type of plant protein and the blending ratio with casein significantly influenced structural organisation and functional performance. Mung bean protein–casein systems exhibited superior stretchability and viscoelasticity compared with hemp protein hybrid systems at equivalent incorporation levels, whereas hemp protein formulations showed greater meltability. These differences were primarily attributed to variations in protein aggregation size and structural composition. Molecular interactions and structural changes were further examined using Raman microspectroscopy and synchrotron-based Fourier transform infrared (S-FTIR) spectroscopy, combined with chemometrics and machine learning approaches. The results showed that mung bean protein–casein systems induced microstructural segregation and changes in β-sheet content, whereas hemp protein–casein systems exhibited greater structural disorder and disulfide bond rearrangement. Increasing casein content reduced plant protein aggregation and promoted a more flexible network, demonstrating that plant proteins are not functionally interchangeable and must be selected according to targeted functionality. Furthermore, mung bean protein was subjected to enzymatic hydrolysis to evaluate physicochemical and structural modifications and to determine whether controlled enzymatic treatment could improve melting functionality in both PCAs and hybrid cheese systems. The results demonstrated that enzymatic hydrolysis effectively reduced the molecular weight and particle size of mung bean protein while increasing surface hydrophobicity, leading to improved meltability in PCA systems. Notably, enzyme specificity exerted a greater influence on functional outcomes than the degree of hydrolysis alone. These results demonstrate that controlled enzymatic modification can tailor functional performance, although optimisation requires balancing solubility, aggregation behaviour, and network continuity. Overall, this PhD thesis demonstrates that the functionality of plant-based and hybrid processed cheese systems is governed by protein-specific interactions, aggregation behaviour, and the resulting network structure. In plant-based systems, different plant proteins generate distinct microstructures that influence texture, meltability, and stretchability. In hybrid cheeses, variations in plant protein–casein interactions lead to different microstructures and corresponding functional outcomes. Enzymatic modification provided an effective means to modulate these interactions and tailor melting behaviour. The findings from this thesis contribute to the rational design of high-plant-protein cheese analogues with improved texture and functional performance.