Effect of the Functionality of Flour and Protein Isolate-Based Ingredients on the Protein Interactions and Structural Properties of Meat Analogue Patties

Meat is a rich protein source, but environmental and health concerns have increased demand for plant-based alternatives. While soy protein isolate dominates the market, issues with allergenicity, GMOs, and monoculture highlight the need for diversified proteins. Legumes such as mung bean and cowpea show strong potential yet remain underexplored, especially in textured forms and regarding their digestibility. This thesis evaluates the physicochemical, structural, functional, and nutritional properties of mung bean, cowpea, and soy in flour and isolate forms, linking ingredient characteristics to texturisation behaviour and digestion. It also examines the ability of legume flours to partially replace SPI and assesses the in vitro digestibility of patties made with mung bean and cowpea isolates, contributing to the development of more sustainable, structurally robust, and nutritionally enhanced plant-based meat analogues. The first experimental chapter provides a comprehensive analysis of mung bean, cowpea, and soy flours and protein isolates, focusing on their potential application in plant-based meat analogues. Mung bean flour (MBF) and cowpea flour (CPF) demonstrated substantial protein content (20.55% and 20.29%, respectively), indicating their potential as rich protein sources as an alternative to soy flour (SBF). CPF exhibited the highest pasting temperature (78.75 °C) and peak viscosity (462.5 cP), reflecting enhanced starch stability and resistance. Among isolates, mung bean protein isolate (MPI) showed the greatest peak viscosity (23.00 cP). Significant differences (p < 0.05) in gelatinisation enthalpy were observed, with CPF displaying the highest values, indicative of stronger starch interactions. Protein profiling identified characteristic bands corresponding to vicilin-like globulins in mung bean (60, 50, 28, and 26 kDa) and globulins in cowpea (48-61 kDa). In soy, β-conglycinin and glycinin subunits were observed at 75, 69, 53, 35, and 17 kDa. Amino acid analysis showed mung bean and cowpea have profiles comparable to soy, providing key essential amino acids and confirming their value as high quality proteins. Mineral profiling revealed flours were rich in magnesium, potassium, and calcium, while isolates contained higher levels of trace minerals such as iron, zinc, and copper. Overall, these findings position mung bean and cowpea as nutritionally robust, underutilised legumes with strong potential as sustainable alternatives to soy in plant-based meat development. Plant proteins are largely globular and lack inherent fibrous structure, so texturisation was applied to enhance their functionality. The second experimental chapter investigated how this process affects mung bean, cowpea, and soy ingredients, evaluating their suitability for producing textured vegetable proteins (TVPs) via low-moisture extrusion for use in meat analogues. The physicochemical and structural attributes of the resulting TVPs were primarily governed by the composition of the raw materials and the extrusion conditions optimised for each type of ingredient, namely mung bean flour, cowpea flour, soy flour, and soy protein isolate. Protein isolate-based TVPs exhibited more defined fibrous structures, higher expansion ratios, and stronger protein-protein interactions, resulting in enhanced water-holding and rehydration capacities. In contrast, flour-based TVPs, particularly textured mung bean flour (T MBF) and textured cowpea flour (T-CPF), demonstrated greater porosity and expansion, with starch components contributing to improved hydration and textural softness compared to that of textured soy protein isolates (T-SPI). While textured soy flour (T-SBF) showed comparatively weaker structural integrity, it retained valuable nutritional attributes. Overall, the findings highlighted the feasibility of using legume-based flours and isolates to partially replace soy protein isolate in plant-based meat formulations. The third experimental chapter examined the physicochemical, structural, textural, and nutritional properties of plant-based patties formulated with textured mung bean, cowpea, and soy flours combined with soy protein isolate. The findings demonstrated that these formulations exhibited competitive protein levels, improved cooking yield, and enhanced 2 moisture retention were observed compared with conventional meat patties and addressed sustainability concerns associated with soy dependency. Incorporating texturised vegetable proteins increased β-sheet structures and strengthened the protein network, leading to improved firmness and cooking stability. Water distribution analysis showed tighter water binding and lower cooking loss than beef patties. Mineral and amino acid profiling confirmed the nutritional viability of the formulations, with good macronutrient and essential amino acid retention, though sulphur-containing amino acids remained limited. PCA further showed that formulations with higher mung bean and cowpea flour closely aligned with the structural and functional attributes of beef and commercial plant-based patties. Overall, textured mung bean and cowpea flours emerged as promising alternatives to textured soy, capable of delivering balanced and high-performing meat analogue systems. The fourth experimental chapter reformulated two promising patty prototypes from Chapter 3, examining the impact of replacing SPI with MPI and CPI in these selected formulations. The reformulated patties exhibited higher protein content, improved cooking yield, and reduced cooking loss, leading to enhanced cooking performance and minimal shrinkage despite the absence of SPI. However, textural analysis revealed reduced hardness, gumminess, and chewiness, making the patties more comparable to beef. These changes resulted from the weaker protein-protein interactions of MPI and CPI compared with SPI, resulting in lower internal binding strength and a softer structure. The limited ability of MPI and CPI to form strong intermolecular bonds and their distinct gelation properties further contribute to these textural modifications. These findings offer valuable insights into the potential of MPI and CPI as alternative protein sources for plant-based meat analogues. The gastrointestinal behaviour of beef, chicken, commercial plant-based patties, and the developed analogues was evaluated using the INFOGEST model, revealing clear structural, physicochemical, and nutritional differences. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 3 confocal imaging, and free amino acid analysis confirmed progressive protein breakdown, with beef and chicken showing efficient hydrolysis and a clear shift to low-molecular-weight peptides. Plant-based patties also degraded but retained more residual peptides, indicating limited enzyme accessibility and the influence of plant anti-nutritional factors. Reduced digestibility in plant analogues was linked to their higher β-sheet content, phytate-mediated mineral and protein binding, and the effects of dietary fibre and flavour compounds, all of which restrict protease access and slow digestion. Overall, this study demonstrates that mung bean and cowpea protein isolates, especially in T CPF formulations, can effectively replace SPI while delivering strong structural and functional performance. Although these plant-based patties showed good cooking and textural properties, they exhibited lower protein hydrolysis than beef and chicken, driven by compact β-sheet structures, anti-nutritional factors, and restricted enzyme accessibility. The work advances theoretical understanding by revealing how flour-based systems with higher starch and fibre undergo distinct thermo-mechanical transitions during extrusion, and by providing mechanistic insights into how legume protein architecture and matrix compactness limit digestibility. Collectively, these findings identify key structural and compositional barriers in legume-based systems and offer guidance for designing plant-based meats with improved texture, functionality, and nutritional bioavailability. This thesis therefore delivers both practical formulation strategies and new scientific insights that extend current knowledge in food protein science.