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Item Fabrication, characterisation, and application of functional protein aggregates derived from faba bean protein isolates : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Auckland, New Zealand(Massey University, 2025-07-14) Hu, YinxuanThis thesis explores the preparation, characterisation, and applications of plant protein aggregates, derived from faba bean protein isolate (FPI). The formation of FPI aggregates was accomplished by various methods, including pH adjustments, salt addition, heat treatment, sonication, and thermosonication (TS). The physico-chemical properties and technofunctional characteristics of FPI aggregates formed by different treatments, such as ζ-potential, solubility, emulsification capability, and particle sizes, were also characterised in this study. Furthermore, the microstructure of the FPI aggregates in solutions was examined using various techniques, including light scattering, microscopies (TEM and SEM), and small angle neutron scattering. Additionally, this project further developed the TS method for formation of FPI fibrillar aggregates at pH 2 and amorphous aggregates at pH 7. The characteristics of FPI aggregates formed by TS and conventional heat treatment (CH) were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography linked to tandem mass spectrometry (LC-MS/MS). In addition, Thioflavin T (ThT) fluorescence, Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD) were applied to investigate the differences in secondary structure between CH-treated FPI and TS-treated FPI, indicating that TS effectively converted FPI structures to be enriched in β-sheets. The gelation behaviours of different FPI aggregates at 10 wt% were studied by examining their rheological properties and observing the microstructure using scanning electron microscopy (SEM), indicating that TS-treatment of FPI at pH 7 facilitated the formation of stronger protein hydrogels. The functionality of FPI aggregates fabricated from various treatments at the oil-water and oil-air-water interfaces was also characterised. Emulsions (O/W) with various oil factions (ϕ) ranging from 0.2 (dilute emulsions) to 0.75 (high internal phase emulsions, HIPEs), were stabilised by suitable FPI aggregates selected based on their different physico-chemical properties. The findings indicate that higher FPI concentrations (~5 wt%) and pH values (~pH 9) result in better emulsification capabilities. Among all FPI aggregates studied in this project, fibrillar aggregates exhibited the best emulsification performance as they could stabilise emulsions with oil content up to 75% (v/v). However, emulsions stabilised by FPI aggregates induced from TS at pH 7 had the greatest application potential due to their long-term stability (up to 28 days) and compatibility with a neutral pH environment. Therefore, another study in this thesis was to investigate the application of FPI aggregates in stabilising vegetable oil-based whipped creams. TS-treated FPI at pH 7 exhibited superior functional properties compared to other treatments, such as CH and ultrasonication (US), in terms of visual appearance, overrun, and stability of whipped cream. Overall, this project provides fundamental insights into the physical-chemical and techno-functional properties of FPI aggregates, including their ability to stabilise and form emulsions, gels, and foams, with an emphasis on their potential applications in innovative food products such as 3D-printed emulsion gels and plant based whipped cream. The enhanced physicochemical and techno-functional properties of FPI aggregates fabricated in this study showed a great application potential as novel food ingredients for formulation of plant-based food products.Item Enhanced properties of non-starch polysaccharide and protein hydrocolloids through plasma treatment: A review(Elsevier B V, 2023-09-30) Sahraeian S; Rashidinejad A; Niakousari MHydrocolloids are important ingredients in food formulations and their modification can lead to novel ingredients with unique functionalities beyond their nutritional value. Cold plasma is a promising technology for the modification of food biopolymers due to its non-toxic and eco-friendly nature. This review discusses the recent published studies on the effects of cold plasma treatment on non-starch hydrocolloids and their derivatives. It covers the common phenomena that occur during plasma treatment, including ionization, etching effect, surface modification, and ashing effect, and how they contribute to various changes in food biopolymers. The effects of plasma treatment on important properties such as color, crystallinity, chemical structure, rheological behavior, and thermal properties of non-starch hydrocolloids and their derivatives are also discussed. In addition, this review highlights the potential of cold plasma treatment to enhance the functionality of food biopolymers and improve the quality of food products. The mechanisms underlying the effects of plasma treatment on food biopolymers, which can be useful for future research in this area, are also discussed. Overall, this review paper presents a comprehensive overview of the current knowledge in the field of cold plasma treatment of non-starch hydrocolloids and their derivatives and highlights the areas that require further investigation.Item The Effect of pH and Sodium Caseinate on the Aqueous Solubility, Stability, and Crystallinity of Rutin towards Concentrated Colloidally Stable Particles for the Incorporation into Functional Foods(MDPI (Basel, Switzerland), 2022-01-14) Rashidinejad A; Jameson GB; Singh H; Papetti APoor water solubility and low bioavailability of hydrophobic flavonoids such as rutin remain as substantial challenges to their oral delivery via functional foods. In this study, the effect of pH and the addition of a protein (sodium caseinate; NaCas) on the aqueous solubility and stability of rutin was studied, from which an efficient delivery system for the incorporation of rutin into functional food products was developed. The aqueous solubility, chemical stability, crystallinity, and morphology of rutin (0.1-5% w/v) under various pH (1-11) and protein concentrations (0.2-8% w/v) were studied. To manufacture the concentrated colloidally stable rutin-NaCas particles, rutin was dissolved and deprotonated in a NaCas solution at alkaline pH before its subsequent neutralisation at pH 7. The excess water was removed using ultrafiltration to improve the loading capacity. Rutin showed the highest solubility at pH 11, while the addition of NaCas resulted in the improvement of both solubility and chemical stability. Critically, to achieve particles with colloidal stability, the NaCas:rutin ratio (w/w) had to be greater than 2.5 and 40 respectively for the lowest (0.2% w/v) and highest (4 to 8% w/v) concentrations of NaCas. The rutin-NaCas particles in the concentrated formulations were physically stable, with a size in the range of 185 to 230 nm and zeta potential of -36.8 to -38.1 mV, depending on the NaCas:rutin ratio. Encapsulation efficiency and loading capacity of rutin in different systems were 76% to 83% and 2% to 22%, respectively. The concentrated formulation containing 5% w/v NaCas and 2% w/v rutin was chosen as the most efficient delivery system due to the ideal protein:flavonoid ratio (2.5:1), which resulted in the highest loading capacity (22%). Taken together, the findings show that the delivery system developed in this study can be a promising method for the incorporation of a high concentration of hydrophobic flavonoids such as rutin into functional foods.Item Characterisation of de-structured starch and its interactions in whey protein isolate gels : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2022) Ang, Cai LingStarch serves as an important additive to enhance the physico-chemical properties of many food products. With the increased pursuit of natural products, there is an increasing demand for “clean-label” starches. In this study, waxy potato starch was physically-modified at elevated temperatures of 120–150 °C for 30 min at 300 rpm, in a pressurised reactor. The treatment converted native starch granules into their macromolecular chains (denoted as de-structured waxy potato starch, DWPS). This doctoral thesis presents the: (i) method of modifying starch (i.e., the de-structuring process), (ii) the mechanism of starch de-structuring, (iii) the rheological changes in DWPS samples and the shear-thickening mechanism, and (iv) the interactions of these DWPS ingredients with whey protein isolate (WPI) in a protein-based gel system, at different pH and ionic strength. The molar mass (Mᵥᵥ), particle size, rheological properties, degree of branching (DB) and side-chain length distribution of DWPS samples were characterised to elucidate the starch de-structuring mechanism. DWPS treated at 120 °C DWPS showed similar Mᵥᵥ (~3.6 × 10⁸ Da) as its native form (~3.7 × 10⁸ Da) indicating that the treatment at 120 °C resulted in the disassembly of starch granules into their macromolecular chains. Reduction in viscosity, Mᵥᵥ and particle size was observed with an increase in temperature from 120 to 150 °C, suggesting a cleavage in amylopectin chains. The DB and side-chain distribution data suggest that the reduction in Mᵥᵥ is likely due to the cleavage at α-1,4 linkages near the middle of the main amylopectin backbone. Particle size analysis by laser diffraction measurements revealed the presence of large fragment particles (> 1 µm) in DWPS samples, indicating that the starch de-structuring process into its macromolecules was incomplete even at 150 °C for 30 min. The DWPS (5% w/w) samples were found to exhibit a wide range of rheological properties—Newtonian, shear-thinning, shear-thickening and anti-thixotropy behaviours—depending on their treatment temperature (120–150 °C). In particular, 120 °C DWPS exhibited interesting shear-thickening, anti-thixotropy and shear-induced gelation. These rheological properties are different from the shear-thinning and thixotropy behaviours observed in most conventionally gelatinised waxy potato starches treated at 95 °C. The complex shear-induced structures of 120 °C DWPS were attributed to a two-step process: (i) upon shear at the critical shear rate (~10–20 s⁻¹), the shear stress caused a size reduction in the starch fragments and (ii) the increased number of small fragments together with the amylopectin chains in very close proximity could lead to the formation of a complex network probably consisting of amylopectin chains and a large number of fragments (2–20 μm). Shear thickening properties were attributed largely to these soft fragment particles colliding and sliding past each other during shear. The data from this study has also shown that the hydrogen bonding, electrostatic, hydrophobic interactions, or the combination of these interactions did not cause the shear-thickening behaviour. The influence of 4% w/w DWPS on 13% w/w WPI gels was studied by characterising the phase stability of the liquid mixtures, and mechanical properties, microstructure, and water-immersion stability of fine-stranded polymeric and coarse-stranded particulate protein gels at pH 7 and pH 5, respectively. At neutral pH, synergistic gel hardness of WPI was obtained with the incorporation of 140 °C DWPS. The increased gel strength was attributed to the enhanced density of a very fine-stranded gel network. The ability of the gel to retain its shape when immersed in water for 40 h was most noticeable for the composite gels containing either gelatinised starch or DWPS samples (swollen gels but with intact shape). In contrast, pure WPI gel and composite gel containing maltodextrin turned into very weak fluid-like and disintegrated gels, respectively. At pH 5, WPI formed particulate gels. The addition of gelatinised starch or DWPS weakened the particulate protein gels, likely due to phase separation and interrupted protein network with starch polymers acting as inactive fillers. The effects of NaCl and CaCl₂ (i.e., type of salts and ionic strength) on the mechanical and microstructural properties of composite gels containing 13% w/w WPI and 4% w/w 140 °C DWPS were also evaluated. Thermodynamic incompatibility between WPI and 140 °C DWPS was observed upon the addition of NaCl (~75 mM) or CaCl₂ (10–75 mM). The combined effects of such thermodynamic incompatibility with the changes in protein connectivity induced by varied ionic strength led to the formation of distinctive gel structures (inhomogeneous self-supporting gels with a liquid centre and weak gels with paste-like consistency) that were different from thermodynamic compatible homogeneous self-supporting gels (pure WPI and WPI + maltodextrin gels). At ≥ 250 mM NaCl, instead of a paste-like texture, a recovered soft self-supporting gel structure was observed when using 140 °C DWPS. The ability to generate a range of textures in WPI gelation-based foods by using 140 °C DWPS under different ionic conditions, is a feasible strategy for structuring high-protein foods for dysphagia—aimed to be either thickened fluids or soft solids. Additionally, this acquired knowledge is also relevant when formulating food gels for 3-D printing. The desirable rheological properties of DWPS samples and their ability to alter WPI gel structure signify the potential of DWPS as a clean-label ingredient to structure foods of specific needs (e.g., whipping cream for enhanced structure upon shear and high-protein foods for dysphagia sufferers).Item Structured emulsion gel systems for delivery of bioactive compounds : 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(Massey University, 2021) Luo, NanThe structure of solid/semi-solid foods greatly impacts on how the food is broken down and digested in the human body, which affects its sensory perception, and the bioaccessibility of nutrients. In this project, heat-set whey protein emulsion gel was used as a model system for solid/semi-solid foods for the delivery of capsaicinoids (CAP); the capsaicinoids were dissolved in the emulsion droplets. The aim was to investigate the effect of emulsion gel structure on the breakdown properties and sensory perception of the gel in human mouth and to understand how gel structure affects its digestion behaviour as well as the release of capsaicinoids during in vitro gastrointestinal digestion. Small and large deformation properties as well as the microstructure of the emulsion gel were evaluated. Eighteen human subjects were used to investigate in vivo oral processing behaviour and sensory perception. The Human Gastric Simulator (HGS) was used for in vitro dynamic gastric digestion and pH-stat for simulated intestinal digestion. Human intestinal epithelial cells Caco-2 were used to evaluate the irritation effect of gastric digesta by the quantification of human interleukin-8 (IL-8) production using enzyme-linked immunosorbent assay (Elisa). Based on the rheological properties, the gels were classified into three groups: semi-solid gel (whey proteins as emulsifier, 10 mM NaCl with d4,3 of ~ 0.2 µm); soft and elastic gels (whey proteins as emulsifier, 10 mM NaCl with d4,3 of ~ 4, 1 and 0.5 µm); hard and brittle gels (whey proteins as emulsifier, 100 mM NaCl with d4,3 of ~ 4, 1, 0.5 and 0.2 µm). Results from in vivo study indicated that the degree of gel fragmentation during mastication was positively correlated with gel hardness (represented by Young’s modulus). A higher degree of fragmentation led to a greater surface exposure during mastication and, therefore, a greater release of capsaicinoid molecules, resulting in greater mouth burn perception. Results from in vitro gastrointestinal digestion of CAP-loaded soft gel and CAP-loaded hard gel showed that the hard gel was disintegrated and hydrolysed slower than the soft gel during gastric digestion. The rate and extent of lipid digestion during intestinal digestion were affected by several factors, such as fat content, gel structure, gel particle size and initial oil droplet size of the gastric digesta. Generally, the soft gel had higher degree of lipid digestion, mainly because of its soft gel structure and lower fat content. The bioaccessibility of CAP was found to be positively correlated with the extent of lipid digestion. The effect of active (whey proteins as emulsifier) versus inactive (Tween 80 as emulsifier) filler particles of CAP-loaded emulsion gels was also investigated. CAP-loaded Tween-80-coated oil droplets were not bound within the whey protein matrix (i.e. emulsion gels containing inactive filler particles) and appeared to be flocculated and heterogeneously distributed in the gel matrix; this led to drastically decreased gel strength. On the other hand, the whey-protein-coated oil droplets had strong interactions with surrounding whey protein matrix contributing to gel strength, and the oil droplets were relatively evenly distributed in gel matrix in CAP-loaded whey protein emulsion gels (i.e. emulsion gels containing active filler particles). During in vivo oral processing, CAP-loaded Tween 80 emulsion gels were readily broken down into small fragments in the mouth at small deformations with less chewing and released large amounts of oil droplets from the gel matrix. In general, the mouth burn perception was positively correlated with degree of gel fragmentation. The large amounts of oil droplets released from the gel matrix during mastication and the inhomogeneous distribution of the oil droplets of the CAP-loaded Tween 80 emulsion gels also contributed to their greater mouth burn perception. During in vitro gastric digestion, the gel with inactive filler particles was disintegrated and emptied out faster than gel with active filler particles, due to its significantly smaller masticated particle size entering the stomach. Large amounts of oil droplets were released during gastric digestion from the gel with inactive filler particles while gel with active filler particles had minor release of oil droplets at the end of digestion. During intestinal digestion, the presence of Tween 80 in gel with inactive filler particles has slowed down the rate and extent of lipolysis, because Tween 80 had certain resistance against replacement by bile salts from the interface. Moreover, the Tween 80 molecules, once displaced by bile salts from the interface, would also participate in the formation of mixed micelles and help solubilize the released CAP molecules, therefore, leading to improved bioaccessibility of CAP. An in vitro method was developed to quantify the gastric irritation of CAP-loaded food formulations during gastric digestion. Results suggest that Caco-2 cells had immune responses to CAP-loaded samples by secreting significant amounts of IL-8, confirming that CAP molecules are inflammatory to Caco-2 cells. The emulsion gel structure was modified using different emulsifiers: whey proteins versus Tween 80. The gastric digesta from CAP-loaded Tween 80 emulsion gel was able to stimulate more IL-8 production than CAP-loaded whey protein emulsion gel. Tween 80 was found to be a proinflammatory factor to Caco-2 cells and could stimulate IL-8 secretion. Overall, this research provided new information on the use of solid/semi-solid systems for delivery of capsaicinoids and how food structure affects disintegration and digestion behaviour and eventually the release of capsacinoids. The outcomes have potential for designing functional foods containing capsaicinoids, with increased incorporation of capsaicinoids in the foods / pharmaceuticals, reduced irritation in the mouth and stomach and increased bioaccessibility in the intestine.