Solubility of chicken breast muscle proteins in solutions of very low ionic strength ($<$0.3 mM) reached 95-98% after certain washing/extracting treatments. Treatment by washing with salt and pH adjusted to neutrality was found critical for complete solubilization. This procedure extracted specific proteins after which the remaining myofibrillar proteins became soluble in water, i.e., ionic strength $<$0.3 mM. SDS-PAGE indicated that these additional proteins were of relatively high molecular mass, $>$66 kDa. Four of the six were tentatively identified as X-protein, C-protein, $\alpha$-actinin and amorphin. These first two proteins are associated with the thick filaments and the latter two with the Z-discs. The other two proteins had molecular masses of 95 and 105 kDa. Those high molecular weight proteins ($>$66 kDa) may restrict the solubilization of the other myofibrillar proteins in water and thus were called the "solubility-inhibiting proteins". At neutral pH, the presence of sodium pyrophosphate and magnesium chloride (10 mM each) and 0.2 M sodium chloride solubilized 57.2 $\pm$ 6.1% of the washed minced chicken breast muscle ("water washed mince") and 60-65% of the myosin. The pyrophosphate may have induced the dissociation of actomyosin into actin and myosin and the presence of magnesium ions may have shifted the isoelectric point of the released myosin toward an acidic pH. These two factors would improve myosin solubility. Solubilization of the myofibrillar proteins, especially myosin, has been considered a prerequisite step for gelation. Good gels could be formed from washed minced chicken breast muscle ("water washed mince") at low (26 mM) or intermediate (150 mM) salt concentrations. The formation of good gels at "low or intermediate" salt levels could be achieved by adjusting the pH of the surimi sample to neutrality prior to heating. The importance of maintaining a neutral pH for gel formation was further demonstrated when gels with low strain values were produced after the pH of the surimi samples previously adjusted to neutrality were brought back to the original low value at 150 mM sodium chloride. At both 26 and 150 mM sodium chloride there was no measurable solubility of myosin. A neutral pH might cause disorganization which might be necessary for gelation. Lack of a relationship between solubility properties and gelation characteristics of the chicken breast myofibrillar proteins were noted under conditions of physiological concentrations or less of salt. Generally, low solubility of myofibrillar proteins did not prevent good gels from forming. Likewise, better solubility did not improve gelation.

A wide variety of food products exist in the form of oil-in-water emulsions. The bulk physicochemical properties and sensory properties of these products is largely determined by the type of emulsifier used to stabilize them. In this study the major factors which influence the properties of oil-in-water emulsions stabilized by whey protein isolate (WPI) are examined. WPI is a value-added ingredient derived from the waste water produced during cheese manufacturing. The properties of emulsions is influenced by the processing and environmental conditions they experience during production, storage and use, for example homogenization, heating, pH, and ingredient interactions. The aim of this study was to examine these factors in a systematic way. During this study it was found that WPI forms stable emulsions above 0.8 wt%, at pH values away from its isoelectric point (pH $<$ 4 or pH $>$ 6) and temperatures below its denaturation temperature (T $<$ 75$\sp\circ$C). The emulsion stability is influenced by the ionic strength, nature of surfactant (e.g. Tween 20, SDS) and polysaccharide (e.g. dextran sulfate) concentration. These changes are induced through modification of the colloidal interactions, interfacial characteristics and/or modification of the aqueous phase. Emulsion stability was monitored through creaming, rheological, light scattering, UV-visible absorption spectroscopic and ultrasonic techniques. The results were interpreted in terms of colloidal interactions, which act between the droplets. The knowledge gained from this research project will enable food manufacturers to improve product quality, reduce manufacturing costs and enhance energy efficiency, by providing a strong scientific foundation for understanding the properties of complex food products.

This work is an investigation of the effects of bed volumes and moisture contents on the textural properties of puff cereals. The first study, two commercial puffed cereals were compressed both individually and in cells of different diameters and heights. Their force-displacement curves were jagged, irregular and irreproducible. The jaggedness of those curves were expressed in terms of their apparent fractal dimension determined from the Richardson plot and the box counting (Kolmogorov dimension) and in term of the mean magnitude of their Fourier power spectrum of their normalized residuals. Three dimensional plots of jaggedness measures versus cell diameter and height produced approximately planar relationship and could be described by the non linear models. Their extrapolation to the individual particle characteristic dimension gave jaggedness values in agreement with those determined experimentally on the individually particle. These results indicated that at least in a certain cereal products, it is possible to assess the brittleness of individual particles from their bulk compressibility pattern. Moreover, this procedure could also be used to estimate the particle stiffness expressed in terms of the fitted force at preselected displacement level (e.g. 15% or 25%) or in terms of the slope of peak force-displacement curve. The second study was moisture content effects on the textural properties of three puffed cereals having various moisture contents. The jaggedness of the force displacement relationship of these cereals was determined in terms of the apparent fractal dimension and the mean magnitude of the power spectrum of their normalized residuals. The plots of these jaggedness measures versus moisture contents yielded a characteristic sigmoid shape very similar to the plots of corresponding sensory crispness or crunchiness versus moisture contents relationships. This close similarity illustrates that the jaggedness measures of the force displacement relationship is manifestation of the same failure events that produce the crispness and crunchiness sensation. In all three products, moisture toughening were detected instrumentally but only in two products, moisture toughening was a clearly corresponding increase in the sensory hardness scores. This indicates that at least in certain cereal products moisture toughening can be perceived simultaneously with brittleness loss.

Lipids in most foods exist as emulsions, therefore it is important to understand the factors affecting oxidative rancidity in emulsions. The ability of iron to promote lipid oxidation in emulsions can be affected by iron's ability to interact with lipids at the emulsion droplet interface. This research was designed to better understand the factors affecting iron interactions with components of emulsions including lipid and surfactant peroxides. Salmon oil-in-water emulsions stabilized by SDS exhibited greater lipid oxidation rates than Tween 20- and DTAB-stabilized emulsions, especially at low pH. Lipid oxidation in salmon oil-in-water emulsions was inhibited by EDTA and the iron-specific chelator, apo-transferrin, indicating that oxidation was promoted by iron. Furthermore, Tween 20-stabilized salmon oil emulsions oxidized faster at pH 7.0 than 3.0. An explanation for this oxidation trend may be due to Fe3+ associating more with Tween 20 emulsion droplets at higher pH's due to its lower solubility. The ability of iron to decompose cumene hydroperoxide in emulsions stabilized by SDS (anionic), Tween 20 (nonionic), and DTAB (cationic) was determined. Peroxide decomposition was greater in SDS-stabilized emulsions due to increased iron-peroxide interactions at the emulsion droplet interface. Factors that decrease iron-emulsion droplet interactions including pH, EDTA, and NaCl inhibited Fe2+ promoted decomposition of cumene hydroperoxide in the SDS-stabilized emulsions. Cumene hydroperoxide in Tween 20 was more susceptible to decomposition by iron at pH 7.0 than 3.0. Polyether surfactants, such as Tween and Brij will accumulate peroxides. Peroxides originating from Tween 20 micelles exhibited changes in peroxide formation and decomposition, the balance of which depended on the concentration and reactivity of iron. In the presence or absence of added iron or copper, peroxides originating from Tween 20 were capable of oxidizing α-tocopherol. Oxidation of α-tocopherol by Fe2+ in Tween 20 micelles was inhibited by EDTA. In conclusion, lipid oxidation rates in emulsions could be lowered by using techniques that decreased the interaction of transition metals at the interfacial region of the droplet.

The influence of high intensity ultrasound during extraction of chitin from North Atlantic Shrimp (Pandalus Borealis) and Fresh Water Prawn (Macrobrachium Rosenbergii) on yield, purity, and crystallinity was investigated. As a subsequent step, effect of ultrasound under different conditions (time, power intensity and temperature) as a pretreatment for deacetylation of chitin from North Atlantic shrimp on yield, purity, time and degree of deacetylation was studied. Yield, mineral and protein content were determined after each processing step. Purity of extracted chitin was assessed as total amount of glucosamine in samples. The crystallinity Index was determined using wide angle X-ray scattering. Analysis of scanning electron microscope images confirmed morphological changes in samples. Degree of acetylation was determined by both Fourier Transform Infrared Spectrometry, (FTIR) and direct titration. Yield of chitin from NAS decreased from 17.02±0.66% to 11.09±1.46% of initial mass with extensive sonication, for FWP the yield decreased from 8.28±1.27 to 5.02±0.64 of initial mass, which was attributed to increased concentrations of depolymerized materials in the wash water. Removal of minerals was not affected by sonication. Application of ultrasound enhanced removal of proteins for NAS from 0.58±0.08% to 0.32±0.03% dry weight and for FWP 0.028±0.06 to 0.013±0.001 dry weight. High Intensity Ultrasound as a pretreatment resulted in accelerated removal of the acetyl-groups under industrial conditions. For samples sonicated for 60 minutes resulted in 90% deacetylated chitosan after 100 minutes of treatment. Application of ultrasound for 15 minutes at 65 and 43 Wcm-2 resulted in DDA of 84.03 and 63.60%, respectively. Samples sonicated at 30°C 65 Wcm-2 for 15 and 30 minutes resulted in 74 and 78% deacetylation of samples. Results were attributed to the chemical and mechanical effect of ultrasound. High intensity ultrasound as a pre-treatment promotes conversion of chitin to chitosan through changes in the chitin structure and by enhancing accessibility of the solvents to its reactive sites. Increased surface area and higher homogeneity of sonicated chitin particles may also be responsible for its increased reactivity. This may results in benefits such as lower production cost, less time and better control of the deacetylation process.

Microbial contamination and oxidation degradation result in unacceptable safety and quality of many foods. Phenolic compounds in essential oils extracted from plant sources may exhibit both radical scavenging and antimicrobial activities. Interest in the use natural phenolic compounds as part of food product formulation has therefore seen a significant increase in order to preserve both food quality and ensure food safety. Despite the high interest in their use, little is known about whether a direct relationship between antimicrobial activity and antioxidant activity of phenolic components exists, i.e. do these components have common characteristic physicochemical properties that allow them to exhibit the two functionalities. Moreover, if there are specific physicochemical properties required for optimal activity in terms of antioxidant and antimicrobial properties, these could be modulated through the design of a suitable delivery system. The objectives of this research were therefore twofold: (1) to determine the relationship between physicochemical properties of phytophenols on lipid oxidation and antimicrobial activity and (2) to develop a delivery polyphenols in complex food system approach to optimize their dual functionalities. To this purpose, model simple phenolic compounds (eugenol and carvacrol) and model complex polyphenolic compounds (polyphenols from cranberries) were characterized in terms of their antimicrobial and antioxidant activities. Results showed both eugenol and carvacrol had remarkably ability to maintain their antioxidant and antimicrobial activities after exposing to oxidative stresses. When complex polyphenolics in cranberries were investigated, CSE powder, a spray dried formulation from cranberry skins, had a higher total phenolic content than 90MX powder, a spray dried formulation from cranberry juice. Also CSE powder simultaneous showed surprisingly high antioxidant and antimicrobial activities. Even more active extracts from CSE powder could be extracted from the powders using solvent extraction. Methanol CSE crude extract had the highest antioxidant and antimicrobial activities compared to water and acetone crude extracts and the CSE powder raw materials compared on the basis of the same concentration of total phenolics. These results were attributed to the affinity of the polyphenolics to the specific solvent resulting in extracts with different polyphenol profiles. The methanol cranberry crude extract (which was the most active extract) was used to formulate an oil-in-water emulsion-based delivery system. Results showed that interfacial composition had a significant effect on stability of emulsions containing the added polyphenols, affecting both antioxidant and also antimicrobial activities. Generally, nonionic surfactants, such as Tween 20, were more capable of formulating stable oil-in-water emulsions that were both oxidatively stable and had antimicrobial activities with cranberry polyphenols. In contrast, emulsions formulated with cationic surfactants were not suitable to produce stable emulsions due to the formation of electrostatically driven complexes with higher interfacial tensions and reduced surface charges. Results of this study thus have offer a new promising method to improve food quality by incorporating simple and complex phenolics in foods that inhibit both lipid oxidation and prevent growth of microorganism.

There was wide variation in hemoglobin levels extracted from muscle tissue of bled and unbled fish. Averaged values indicated that bleeding removed 23–44% of the hemoglobin. This suggested that the residual blood level in muscle of bled fish was high. There also was a wide variation in sensory scores and TBARS values from bled and unbled fish stored at 2°C. Bleeding significantly reduced rancidity in minced trout whole muscle, minced mackerel light muscle, intact mackerel dark muscle, but not minced mackerel dark muscle stored at 2°C. The reduction was in the number of fish that had longer shelf-life; the shelf-life of some bled fish was not improved. Hemoglobin levels in tail-bled and gill-bled trout were not significantly different. Evidence of hemoglobin subunits in extracts was observed. Hemoglobin could account for all of the lipid oxidation capacity of whole blood added to washed cod muscle. The evidence suggested that the active form of hemoglobin had the heme iron intact. When a critical level of hemoglobin was present, breakdown of lipid hydroperoxides occurred that was accompanied by oxidation odor. At a low level of preformed lipid hydroperoxides (3.6 μM), reduced hemoglobins stimulated lipid hydroperoxide formation, whereas methemoglobin did not. This suggests that reduced hemoglobins played an important role in the early stages of lipid oxidation. Lowering pH in the range of 7.6 to 6.0 greatly accelerated lipid oxidation induced by reduced hemoglobins. The sharp decrease in hemoglobin oxygenation in this pH range suggested a possible role for deoxyhemoglobin. Hemoglobin autoxidation also occurred more rapidly at pH 6.0 compared to 7.2. Methemoglobin promoted oxidation of washed cod lipids equally at pH 6.0 and 7.6. Increasing the level of preformed lipid hydroperoxides activated methemoglobin as a catalyst when linoleic acid was the lipid substrate. In addition to increased pH, blood plasma, ascorbate, and prevention of erythrocyte lysis inhibited hemolysate-induced lipid oxidation. The water-extractable fraction of cod muscle was highly inhibitory at pH 6.7. Finally, adding in-situ levels of trout blood to reduced-lipid washed cod caused strong rancid odor to develop during 2°C storage in samples containing no more than 0.1% lipid.

Solubilization of fish myofibrillar proteins at low ionic strength, neutral pH was found to be species dependent and possibly linked to pH of the intact muscle. Two distinct groups formed, one soluble, the other non-soluble, depending upon their initial muscle pH. Incubation of Atlantic cod muscle, whose proteins were initially soluble, in low pH (5.8–6.2) adjusted media resulted in a reversible, time-related insolubilization. Adjustment of pH to slightly greater than neutral (7.2) in Atlantic mackerel light muscle, a nonsolubilizing specie resulted in the almost complete solubilization of its myofibrillar proteins. Most species investigated achieved complete solubilization of their myofibrillar proteins at low ionic strength, pH 3.0. Gadoid fish that underwent abusive storage were insoluble and had moderately high levels of dimethylamine possibly indicative of protein crosslinking. Centrifugation of acid soluble proteins removed a substantial portion of membrane lipids as determined by phosphorus detection. Recovery of acid solubilized proteins using isoelectric precipitation resulted in yields around 85%. High yields were due to the recovery of sarcoplasmic proteins in addition to myofibrillar proteins. Protein isolates from fish and chicken breast muscle were found to retain functionality, producing gels at neutral pH with high stress and strain values. Dynamic rheological testing found that acid solubilized gels begin formation at a lower temperature than gels that consistently stayed at neutral pH. At low protein concentration, membrane removal decreased the storage modulus of the gel. Membrane removal was found to improve stability to oxidation in Atlantic mackerel, however, long term stabilization may require an antioxidant system. Freeze-dried protein isolates were prepared from acid solubilized fish proteins, when re-hydrated, produced gels of good color and acceptable strain and stress.

Mass transport processes in food emulsions were investigated by low intensity ultrasonic spectroscopy and optical profiling techniques, e.g., droplet creaming and small molecule diffusion. Gravitational separation of droplets was observed in non-flocculating and flocculating oil-in-water emulsions. Flocculation was induced by either a depletion mechanism or by electrostatic screening. The creaming velocity of oil-in-water emulsions stabilized by a non-ionic surfactant is strongly influenced by droplet flocculation and droplet size and concentration. The creaming velocity increased with droplet flocculation because of the increase in particle size, and decreased with droplet concentration due to particle crowding effects. The creaming velocity of flocculated emulsions could be approximately modeled using equations developed to describe creaming in non-flocculated systems, provided the increase in the effective volume fraction due to flocculation was taken into consideration. The diffusion of small molecules, such as sugars, into a biopolymer gel and a colloidal dispersion was studied in a xanthan solution and an oil-in-water emulsion. It was found that ultrasonic profiling is an efficient and useful method for non-destructively monitoring molecular diffusion in optically opaque biopolymer solutions and emulsions. The biopolymer tested (1% xanthan) had little influence on the diffusion coefficient of sucrose in aqueous solutions because the sugar molecules movement through the biopolymer network was not significantly retarded. Oil droplets of up to 20 wt% a caused slight decrease in the diffusion coefficient of sucrose through oil-in-water emulsions, which was in agreement with theory and previous studies of molecular diffusion in colloidal dispersions. All studies were conducted with both non-destructive analytical techniques; ultrasonic spectroscopy and optical profiling. It is essential to prove that monitoring the kinetics of mass transport on a single sample is time and cost efficient. It was found that ultrasonic profiling spectroscopy had major advantages for studies on; on optically opaque systems, whereas optical profiling was more sensitive for studies on optically transparent systems. The application of both techniques on model systems and real foods were demonstrated.

The trace elements iron (Fe), copper (Cu) and zinc (Zn) play important roles in neuronal differentiation, function, and survival. These metals have been implicated in neuropsychiatric and neurodegenerative disorders, such as Parkinson's, Alzheimer's, and Wilson's diseases, that have been associa ted with changes in neuronal proliferation and differentiation, and are characterized by apoptotic neuronal death. The work described in this dissertation explores the molecular roles of Fe, Cu, and Zn, with a particular focus on gene expression during neuronal differentiation and apoptosis. The first part of this work uses both in vivo and in vitro models to explore the role of Fe in neuronal differentiation. This work showed that retinoic acid, a known molecular regulator of neuronal differentiation, may act through its ability to increase expression of ferritin H, a protein that plays a role in neuronal Fe utilization. The second part of this work examined the role of Cu in the molecular mechanisms responsible for neuronal apoptosis. This work showed that Cu toxicity, such as that seen in several neurodegenerative disorders, induces neuronal apoptosis that is dependent on the expression and nuclear translocation of the tumor suppressor protein p53. Because p53 acts as a DNA-binding transcription factor, fo llow-up work used oligonucleotide array to identify p53-target genes that regulate neuronal survival and apoptosis. Both pro-apoptotic genes (IGFBP-6 and c-jun) and anti-apoptotic genes (Hsp 70 and 27) were regulated by p53 under conditions of neuronal Cu-overload. We also showed that p53 trafficking was regulated by Zn. High concentrations of neuronal Zn prevented nuclear translocation of p53, inhibiting it functions in gene transcription and apoptosis. The final part of this work used subtracted cDNA libraries, differential hybridization, and high throughput gene expression profiling (microarray) to identify new molecular roles of Zn in the olfactory bulb, a region of the brain known to have high concentrations of Zn and an association with several neurological disorders. This work showed that Zn regulates a variety of bulb genes involved in both neuroplasticity and apoptosis, and suggests that previously reported positive effects of caloric restriction on neuronal survival and plasticity are abrogated by the development of Zn deficiency. In summary, this work has identified a number of novel molecular roles for the trace elements Fe, Cu, and Zn in neuronal differentiation and apoptosis, and suggests a role for these metals in neurological disorders associated with changes in neuronal survival. / A Dissertation submitted to the in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2004. / February 23, 2004. / Apoptosis, Ferritin H, Zinc, Copper, Gene, P53 / Includes bibliographical references. / Cathy W. Levenson, Professor Directing Dissertation; Charles Ouimet, Outside Committee Member; Marc Freeman, Committee Member; J. Michael Overton, Committee Member; Paul Trombley, Committee Member.