Due to the limited number of approved antioxidants for food applications, several alternative strategies to improve antioxidant performance have been developed by focusing on synergistic antioxidant interactions. Susceptibility to lipid oxidation in food systems is the result of the summation of antioxidative and prooxidative mechanisms. Understanding the sometimes paradoxical behavior of antioxidants and prooxidants is a vital key to design synergistic antioxidant systems suitable for particular foods. This research focused on 3 main strategies to improve the performance of antioxidant activity in oil-in-water emulsions. The first part of this research has been focused on inhibition of lipid oxidation by a combination of the modification of liposomal surfaces by chitosan-coating techniques along with addition of rosmarinic acid esters of varying polarity. Repelling metal ions away from the interface of positively charged liposomes can inhibit lipid oxidation (induced by Fe2+), and also reduce antioxidant loss by Fe3+ reduction. As a result, lipid oxidation can be inhibited synergistically because of a reduction in the prooxidant activity of iron. Second, understanding non-linear antioxidant behavior (the cut-off effect) of antioxidant esters in oil-in-water (O/W) emulsions was also studied to determine how the distributions and locations of antioxidants impacted their antioxidant activity. Antioxidant activity of rosmarinic acid was improved by esterification with alkyl chain lengths between 4 to 12 carbons due to increased ability to partitioning at the interface in oil-in-water emulsions. Surfactant micelles which could increase or decreased the concentration of the antioxidants at the emulsion droplet interface altered antioxidant activity. In the last part of this research, rosmarinic acid and its esters were found to be an excellent tool for studying how antioxidant location could impact its ability to interact with α-tocopherol in O/W emulsions. Synergistic, additive, and antagonistic effects were observed in the combinations between the rosmarinate esters with α-tocopherol. Increases in alkyl chain lengths of rosmarinic acid have influenced both the partitioning of the rosmarinate esters as well as their ability to they interact with α-tocopherol at the interface of oil-in-water emulsions. Fluorescence quenching and EPR studies showed that water soluble rosmarinic acid (R0) exhibited more interactions with á-tocopherol than any of the esters (R4-R20). Synergistic antioxidant interactions between rosmarinic acid and α-tocopherol could not be explained by electron transfer mechanisms, but formation of caffeic acid from rosmarinic acid. Due to the thermodynamic infeasibility and the fact that increases in α-tocopherol degradation rates, α-tocopherol could not be regenerated efficiently by rosmarinic acid. This formation of caffeic acid was proposed to be responsible of the synergistic activity of R0 and α-tocopherol since the formation of an additional antioxidant could further increase the oxidative stability of the emulsion.

Filled hydrogel particles consisting of emulsified oil droplets encapsulated within a hydrogel matrix were fabricated based on the phase separation of proteins and polysaccharides through aggregative and segregative mechanisms. A 3% (wt/wt) pectin and 3% (wt/wt) caseinate mixture at pH 7 separated into an upper pectin-rich phase and a lower casein-rich phase. Casein-coated lipid droplets added to this mixture partitioned into the lower casein-rich phase. When shear was applied, an oil-in-water-in-water (O/W1/W2 ) emulsion consisting of oil droplets (O) contained within a casein-rich dispersed phase (W1) suspended in a pectin-rich continuous phase (W2) was formed. Acidification from pH 7 to 5 promoted adsorption of pectin onto casein-rich W1 droplets, forming filled hydrogel particles. Particles were then cross-linked using transglutaminase. Particles were assessed for stability to changes in pH, increasing levels of salts (sodium chloride and calcium chloride), and susceptibility to lipid oxidation. Both cross-linked and not cross-linked particles were stable at low pH (pH 2-5). At high pH, cross- linked particles maintained their integrity while not cross-linked particles disintegrated. Particles were stable to sodium chloride (0-500 mM). Calcium chloride levels above 4 mM resulted in system gelation. The rate of lipid oxidation for 1% (vol/vol) fish oil encapsulated within filled hydrogel particles was compared to that of oil-in-water emulsions stabilized by either Tween 20 or casein. Emulsions stabilized by Tween 20 oxidized faster than either filled hydrogel particles or casein stabilized emulsions, while filled hydrogel particles and casein stabilized emulsions showed similar oxidation rates. Using an in-vitro digestion model, the digestion of lipid encapsulated within filled hydrogel particles was compared to that of a casein stabilized oil-in-water emulsion. Results showed similar rates of digestion for both hydrogel and emulsion samples. Attempts to fabricate particles using free oil (rather than emulsified oil) were unsuccessful and resulted in the formation of large non-encapsulated oil droplets (d ~10 &mgr;m). By controlling particle concentrations of biopolymer, water, and oil, it was possible to fabricate particles that were highly resistant to gravitational separation which was attributed to the equivalent density of the continuous and particle phases. Results highlight the potential applications and versatility of this delivery system.

The aim of this thesis was to develop and test novel food-grade nanodispersions, such as nanoemulsions and solid lipid nanoparticles, for the encapsulation, protection and delivery of bioactive lipophilic food components. Initially, the impact of system composition and homogenization conditions on the formation of nanoemulsions using a high pressure homogenizer (microfluidizer) was examined. The mean particle diameter decreased with increasing homogenization pressure and number of passes, with a linear log-log relationship between mean particle diameter and homogenization pressure. Surfactants emulsifiers formed smaller droplets than protein emulsifiers, which was attributed to their ability to rapidly adsorb to the droplet surfaces during homogenization. At low oil phase-to-aqueous phase viscosity ratios, much smaller mean droplet diameters could be achieved for SDS (d ∼ 60 nm) than for β-lactoglobulin ( d ∼ 150 nm). The effectiveness of various biopolymer emulsifiers at forming and stabilizing model beverage emulsions was examined: β-lactoglobulin (BLG); gum arabic (GA); modified starch (MS). Orange oil-in-water nanoemulsions (5% oil) were prepared using high pressure homogenization. Extensive droplet aggregation occurred in BLG-stabilized nanoemulsions around their isoelectric point, at high salt concentrations, and at high temperatures, due to changes in electrostatic and hydrophobic interactions. There was little effect of pH, ionic strength, and temperature on emulsions stabilized by GA or MS, due to strong steric (rather than electrostatic) stabilization. The potential of utilizing oil-in-water (O/W) nanoemulsions stabilized by a globular protein (β-lactoglobulin) for encapsulating and protecting β-carotene was examined. The influence of temperature, pH, ionic strength, and emulsifier type on the physical and chemical stability of β-carotene enriched nanoemulsions was investigated. The rate of color fading due to β-carotene degradation increased with increasing storage temperature (5 to 55 ºC), decreasing pH, and was largely independent of ionic strength (0 to 500 mM of NaCl). β-carotene degradation was considerably slower in β-lactoglobulin-stabilized nanoemulsions than in Tween 20-stabilized ones. The rate of β-carotene degradation decreased upon addition of additional antioxidants. EDTA was found more effective than ascorbic acid, and Coenzyme Q10 was more effective than vitamin E acetate. The utilization of water-soluble and oil-soluble antioxidants in combination (EDTA and vitamin E acetate) was less effective than using them individually. Solid lipid nanoparticles (SLN) were prepared by homogenizing at a temperature (≈ 80°C) exceeding the melting point of the lipid phase (tripalmitin), and then cooling the resulting oil-in-water nanoemulsions to induce lipid droplet crystallization. Blending tripalmitin with low melting point lipids (either medium chain triglycerides or orange oil) prior to homogenization led to a considerable alteration in the phase behavior and stability of SLN. The presence of the carrier oils reduced the crystallization temperature, melting temperature, and melting enthalpy of tripalmitin. The bioaccessibility of β-carotene encapsulated within nanoemulsion-based delivery systems was examined. A non-ionic surfactant (Tween 20) was used as an emulsifier and long chain triglycerides (LCT), medium chain triglycerides (MCT) or orange oil were used as carrier oils. The bioaccessibility of β-carotene was negligible (≈ 0%) in orange oil nanoemulsions because no mixed micelles were formed to solubilize β-carotene, and was relatively low (≈ 2%) in MCT nanoemulsions because the mixed micelles formed were too small to solubilize β-carotene. In contrast, β-carotene bioaccessibility was relatively high (about 66%) in LCT nanoemulsions because the mixed micelles were large enough to solubilize the bioactive molecule. Overall, our results have important implications for the design of effective delivery systems for encapsulation of carotenoids and other lipophilic bioactive components so that they can be incorporated into functional food and beverage products.

The increase in obesity and overweight in many countries has led to an upsurge of interest in the development of reduced fat food products. However, the development of these products is challenging because of the many roles that fat droplets normally plays in these food products, including contributing to flavor, texture, appearance, and bioactivity. The goal of this research was to develop novel reduced-fat emulsions based on hetero-aggregation of oppositely charged food−grade colloidal particles or polymers. Initially, lactoferrin (LF) and β-lactoglobulin (β−Lg) were selected as emulsifiers to form protein-coated fat droplets (d43 ∼ 0.38 μm) with opposite charges at neutral pH: pKaβ−Lg ∼ 5 < pH 7 < pKaLF ∼ 8.5. Droplet aggregation occurred when these two emulsions were mixed together due to electrostatic attraction. The structural organization of the droplets in these mixed emulsions depended on the positive-to-negative particle ratio, particle concentration, pH, ionic strength, and temperature. The nature of the structures formed influenced the rheology, stability, and appearance of the mixed emulsions, which enabled some control over emulsion functionality. The largest microclusters were formed at particle ratios of 40% LF−coated and 60% β−Lg−coated fat droplets, which led to mixed emulsions with the highest apparent viscosity or gel strength. At low total particle concentrations (0.1%), there was a relatively large distance between microclusters and the mixed emulsions were fluid. At high particle concentrations (>20%), a three−dimensional network of aggregated droplets formed that led to gel−like or paste-like properties. The influence of environmental stresses on the physicochemical stability of the microclusters formed by hetero−aggregation was investigated: pH (2−9); ionic strength (0−400 mM NaCl); and temperature (30−90 ºC). Large microclusters were obtained at pH 7 (d43 ∼ 10 μm) with the absence of salt at room temperature. More acidic (< pH 6) or alkaline (> pH 8.5) solutions resulted in smaller aggregates by minimizing the electrostatic attraction between the protein-coated fat droplets. Microclusters dissociated upon addition of intermediate levels of salt, which was attributed to screening of attractive electrostatic interactions. Heating the microclusters above the thermal denaturation temperature of the proteins led to an increase in gel strength, which was attributed to increased hydrophobic attraction. The influence of hetero-aggregation of lipid droplets on their potential biological fate was studied using a simulated gastrointestinal tract (GIT). Results showed that the mixed emulsions had high viscosity in the simulated oral environment but exhibited similar rheological properties and particle characteristics as single-protein emulsions in the simulated gastric and small intestinal tract regions. The mixed emulsions also had similar lipid digestion rates in the simulated small intestine as single-protein emulsions suggesting that they could be used as delivery systems for bioactive lipophilic compounds in reduced fat food products. The possibility of using more practical food ingredients to promote hetero-aggregation system was also examined. Whey protein isolate (positive) and modified starch (negative) were selected as building blocks due to their opposite charges at pH 3.5. The largest aggregates and highest viscosities occurred at a particle ratio of 70% MS and 30% WPI, which was attributed to strong electrostatic attraction between the oppositely charged droplets. Particle aggregation and viscosity decreased when the pH was changed to reduce the electrostatic attraction between the droplets. Finally, the influence of interfacial properties on the chemical stability of bioactive components in emulsion-based delivery systems containing mixed proteins was studied. Lactoferrin (LF: pI ∼ 8) and β-lactoglobulin (β−Lg: pI ∼ 5) were selected to engineer the interfacial properties. Interfaces with different structures were formed: LF only; β-Lg only; LF-β−Lg (laminated); β−Lg−LF (laminated); β−Lg /LF (mixed). The influence of pH, ionic strength, and temperature on the physical stability of β-carotene-enriched emulsions was then investigated. LF- emulsions were stable to the pH change from 2 to 9 but the aggregation was occurred in intermediate pH for other emulsions. β−Lg− emulsions aggregated at low salt concentration (≥ 50mM NaCl), however other emulsions were stable (0 − 300mM NaCl). β−Lg /LF (mixed) emulsions were unstable to heating (≥ 60 ºC), but all other emulsions were stable (30 to 90 ºC). Color fading due to β−carotene degradation occurred relatively quickly in β−Lg− emulsions (37 ºC), but was considerably lower in all other emulsions, which was attributed to the ability of LF to bind iron or interact with β-carotene. Overall, this study shows that hetero-aggregation may be a viable method of creating novel structures and rheological properties that could be used in the food industry.

Rapid detection technologies with high sensitivity and selectivity for pathogenic bacteria are critical in food safety and quality assurance. Traditional laboratory benchtop techniques (i.e. culture and colony counting) are time consuming and require complex sample handling. Microfluidics-based Lab-on-a-Chip (LOC) systems offer a detection alternative where all detection steps are on one portable miniaturized device. The miniaturization of rapid foodborne pathogen detection at a low cost is especially ideal for resource-limited settings or for field-use. The goal of this study was to develop a disposable miniaturized microfluidic device for on-site pathogen detection. Capillary-driven microfluidics have been introduced in this study. Compared to traditional microfluidics which rely on external devices for operation, our capillary-driven biosensors are self-priming microfluidics in which fluid flowing is actuated by surface wetting. Polymer substrates are used instead of standard silicon-or glass- based materials, due to the ease of microfabrication and their low cost for disposable applications. Two fabrication methods for polymeric microfluidic chips were developed. A plastic replication technique by hot embossing was developed in order to produce plastic prototypes from a master made from copper and photoresist (SU-8). The second rapid prototyping method included laser ablation and adhesives-bonding was developed. Electronic components have been developed using inkjet-printing techniques, due to their low cost and ease of design. The inkjet-printed silver and gold electrodes have been used in enzyme-based electrochemical biosensors in this study. The On-chip Electrowetting (EW) Valve concept was incorporated, for the sequential delivery of reagents to the reaction site in a miniaturized capillary-driven microfluidic biosensor. The valve could be actuated at a low voltage (4.5 V) and was realized on various substrates. Bacteriophage-based detection is used for pathogenic bacteria, due to the fact that phages can be extremely host-specific and phage-based detection allows the differentiation between live and dead bacteria. Immunomagnetic separation (IMS) was used for sample pre-incubation and rapid separation. As a result, different capillary-driven microfluidic platforms have been developed for various applications, such as chemiluminescence detection for nucleic acid target, colorimetric immunoassay as well as electrochemical detection of T7 bacteriophage.

Freezing and thawing are important food processing operations, often carried out by placing objects in an air stream which exchanges heat with the objects. These objects, even when rectangular, have more than two surfaces and the heat transfer coefficient (h) for each surface is almost always different. Based on equations for predicting effective heat capacities, enthalpies and thermal conductivities, freezing and thawing equations for time-temperature estimation for an isolated infinite slab and low Biot number (Bi) have been developed. These were extended using correction factors based on Bi, as to provide equations which can be used for rectangular objects in a row with two, or four exposed faces. Because thermal conductivity (k) changes during freezing and thawing, Bi also changes. An equation for the variation in Bi as k varies is obtained. Average Bi values defined by 2ha/(k(,o)+k(,f)), 0.5 ha/k(,o)+0.5 ha/k(,f), and ha/k(,o) for thawing and ha/k(,f) for freezing were also used. When h is different on opposite surfaces, the maximum temperature does not occur at the center plane of the object. Approximated solutions are obtained using correction factors based on Bi values determined with the position of the plane where the maximum temperature occurs or based on the different Bi values obtained with the different h. Average temperature vs time results are compared using these Bi and the numerical PDE solutions. h, was experimentally evaluated and an alternative correlation was developed. With this correlation the individual surface h's could be predicted as a function of the approach air velocity and the geometric characteristics of the row and object, and the freezing and thawing temperature vs. time behavior of the object placed in the row could then be predicted using this h. Typical cases were tested and the differences between the experimental results, the values predicted by the approximation equations for low Bi and the numerical solutions of the PDE were determined.

Nutritional oil-in-water emulsion products are usually fortified with minerals, which can interact with other ingredients and cause physiochemical instability reducing product shelf-life. The objective of this study was to investigate the impact of mineral-ingredient interactions on properties and stability of protein stabilized emulsion in order to find potential approaches to prevent the emulsion instability. Commercial nutritional beverages had mean particle diameters between 0.5–11 μm and pH values around 7. The monovalent and divalent minerals present in the products at the highest average concentrations were potassium and calcium, respectively. Both calcium and potassium ions induced droplet aggregation in a model emulsion, which had a mean particle size of 0.6–0.7 μm, pH value at 7, 7% oil, and was stabilized by whey protein isolate (WPI). The aggregation led to an increase in emulsion particle size, apparent viscosity, shear-thinning behavior, and creaming instability with increasing mineral concentration. The critical concentration of the aggregation was 3–7 mM for calcium and 200 mM for potassium. The emulsion particle surface charge was decreased by minerals, probably because of electrostatic screening and binding of the mineral ions to negatively charged adsorbed proteins. Calcium binding affinity and enthalpy of EDTA was greater than those of citrate and WPI, respectively. EDTA and citrate bound calcium ions in 1:1 ratio, whereas WPI bound to calcium ions in about 1:3 to 1:4 ratio. WPI that was heated at 70–90°C had an increase in calcium binding affinity and enthalpy. Citrate consisted of two types of binding sites. EDTA, citrate, and non-adsorbed WPI prevented or reduced calcium-induced droplet aggregation with increasing their concentration. EDTA was more effective than citrate and WPI, respectively. The chelating agents lowered free-calcium concentration and magnitude of particle surface charge. The emulsions had less free-calcium ions to induce the instability. Heat treatment of non-adsorbed WPI at 70 or 90°C did not improve the performance of non-adsorbed WPI. EDTA, citrate, and non-adsorbed WPI could not prevent droplet aggregation induced by thermal treatment, when emulsions containing calcium were heated at 80–120°C for 15 min. It was suggested that hydrophobic attraction upon heating induced droplet aggregation.

The concentrations of ascorbate, glutathione, $\alpha$-tocopherol, ubiquinone-10, and ubiquinol-10 and their rates of loss in the light and dark muscle of Atlantic mackerel were determined. This was done for the purpose of determining their usefulness as indices of postmortem age and shelf-life quality. A paired fillet technique was used on groups of six fish, freezing one fillet immediately at ${-}62\sp\circ$C as a time "0" control and storing the other on ice for 0.5, 1, 2, 4, 7, or 10.7 days, and then freezing it at ${-}62\sp\circ$C until assayed. Sensory evaluations and determination of thiobarbituric acid-reactive substances (TBARS) were done on the same fish at the same storage times used for antioxidant determinations. All of the antioxidants tested were lost more rapidly in the dark muscle than in the light muscle. In dark muscle, ubiquinol-10 and glutathione were lost most rapidly, followed by ascorbate, which was lost approximately twice as fast as $\alpha$-tocopherol, which in turn was lost two to three times faster than ubiquinone-10. In light muscle, ascorbate and glutathione were lost at approximately the same rate, which was twice as fast as for $\alpha$-tocopherol which was again twice as fast as ubiquinone-10. The low concentration of ubiquinol-10 in light muscle decreased very slowly. Ascorbate, glutathione, and $\alpha$-tocopherol could be used as indices of freshness for the early changes in sensory quality in light muscle, while all of the antioxidants were early indicators of sensory change in dark muscle. Odors indicative of rancidity were detected by the fourth day of storage in dark muscle and the seventh day in light. Polynomial equations were derived to describe the relationship of sensory scores and antioxidant loss in light and dark muscle. The effect of time of frozen storage was determined by comparing time "0" fillets stored for up to 62 days at ${-}62\sp\circ$C. Ascorbate and glutathione decreased significantly (p $<$ 0.01) in light muscle while glutathione decreased and ubiquinol-10 increased significantly (p $<$ 0.01) in dark muscle between the first and fifth days of frozen storage. Ubiquinone-10 demonstrated antioxidant activity in a model system containing 2,2$\sp\prime$-azobis(2,4-dimethylvaleronitrile) (AMVN).

Structural, thermo-mechanical and molecular changes in starch gels and bread were studied at variable moisture contents, storage times and temperature. Glassy-rubbery transition was characterized in systems with and without "freezable" water using thermal analysis and NMR (Nuclear Magnetic Resonance). Dynamic Mechanical Analysis (DMA) in conjunction with Differential Scanning Calorimetry (DSC) were used to describe structural relaxation (long range) while $\sp1$H cross-relaxation NMR and $\sp{13}$C CP-MAS (Cross-Polarization/Magic Angle Spinning) NMR were used to observe the starch molecular motions (short range). DMA results showed a transition region covering a large temperature range which was found to be moisture dependent. Overlapping tan $\delta$ (T) curves were deconvoluted into asymmetric double Sigmoidal curve (glassy-rubbery transition) and Gaussian curve (ice melting). The latter was found to highly correlate with the amount of "freezable" water (DSC data). The asymmetric curve was also confirmed as a glassy-rubbery transition on a molecular level using $\sp1$H cross-relaxation NMR and $\sp{13}$C CP-MAS NMR. Both NMR data showed a decrease in starch mobility with lowering temperature around the asymmetric (glassy-rubbery) transition observed by DMA. As the moisture content decreased so that the amount of "freezable" water was depleted, the transition temperature range increased, shifting to a higher temperature, typical of a glassy-rubbery transition. Firming of aging starch gels was related to amylopectin recrystallization (which followed amylose crystallization), glassy-rubbery transition or network formation (DMA), starch molecular mobility (NMR) and "freezable" water. A distribution of glassy-rubbery transition temperatures increased with storage time. The structural network was nonuniformly distributed leading to heterogeneous domains exhibiting different glassy-rubbery transition temperatures. However, only increased overall solid fractions (intensity) was observed by both $\sp1$H cross-relaxation and $\sp{13}$C CP-MAS NMR methods but no change in the molecular transition temperature range. This suggested that the "network" formation formed during the aging of starch did not occur on the molecular level but on a structural one.

The objective of this research was to study the radiolysis of polyunsaturated lipid systems and to provide data for the development of irradiation detection methodology for seafood. Polyunsaturated triacylglycerols and methyl esters, and mackerel lipids were irradiated with $\gamma$-rays from $\sp{60}$Co at ambient temperature. Radiolytic compounds were collected, fractionated, and identified using vacuum distillation, gas chromatography, and mass spectrometry. In large part the same major radiolytic hydrocarbons produced from the saturated fatty acids, i.e. $\rm C\sb{n-1},\ C\sb{n-2+db},\ C\sb{n-1+db},\ and\ C\sb{n-2},$ were also produced from the polyunsaturated systems suggesting that the primary events are the same, i.e. preferential scission at sites near the ester carbonyl producing specific alkyl, acyl, acyloxy, and acyloxy-methylene free radicals. Several polar compounds including the methyl and ethyl esters, cyclobutanones and other ringcontaining structures have also been identified as radiolytic products from polyunsaturated systems. The yield of radiolytic products from polyunsaturated chains was significantly less than that from saturated systems, suggesting a reduced cleavage along the fatty acid chain due to the stability of C=C and bonds alpha to these, while cleavage of C-H bonds beta to unsaturated linkages are favored leading to non-volatile products. The large number of strictly radiolytic compounds provides sufficient data for the development of radiation detection methodology for seafoods.