Alterations in the protein and lipid components of lean fish species were studied to elucidate the nature of protein denaturation in Gadus morhua and Melanogrammus aeglefinus during frozen storage. Frozen storage of lean fish, Gadus morhua and Melanogrammus aeglefinus led to the formation of ice crystals, which contributed to protein denaturation. Ice crystals were larger in fish fillets stored at -10°C compared to matching fillets stored at -30°C as studied by light microscopy at -20°C, which indicated damaged muscle fibre by compression. Protein denaturation was also attributed to the effect of lipid oxidation products. The presence of oxygen in the muscle system and high, hydrolytic enzyme and lipoxygenase activity led to increased free fatty acids and lipid oxidation respectively. The peroxide value (PV), conjugated dienes, thiobarbituric acid reactive substances (TEARS) and olefinic to aliphatic protons ratio by 1H NMR spectroscopy was indicative of oxidative deterioration of the lipid components of Gadus morhua and Melanogarmmus aeglefinus during frozen storage especially at -10°C. The ratio of the C=C to the aliphatic groups as assessed by FT-Raman spectroscopy also decreased progressively over the frozen storage period. The 1H NMR, conjugated diene and FT-Raman spectroscopic measurements were found to be effective and less labour intensive techniques for finger printing lipid oxidation than traditional methods. Intact muscle analysis using differential scanning calorimetry (DSC) showed that protein components of the muscle were denatured resulting in altered Tm and DeltaH values. FT-Raman spectroscopy of fish tissue confirmed changes in the proteins and showed decreased levels of alpha-helix and increased ?beta-sheet content (%) as well as changes in hydrophobic groups after frozen storage. These changes were pronounced in samples stored at -10°C compared to samples stored at -30°C. Model systems of protein-lipid complexes were studied using DSC, FT-Raman spectroscopy and ELISA. The effect of lipids and the primary and secondary oxidation products altered the conformation of myosin, collagen and water soluble proteins during freezing and frozen storage. DSC parameters namely Tm and DeltaH values indicated the degree of denaturation of fish proteins, when frozen in the presence and absence of lipids. It is proposed that ice crystal formation resulted in the removal of the hydration shell of the proteins and the overall rearrangement of the stabilising forces; this allowed protein-lipid interaction to take place and induced further protein denaturation. Reduced immune affinity of the myosin-lipid systems towards the myosin antibody compared to the control native myosin indicated conformational changes of the myosin molecule. The addition of lipids (DHA, EPA, extracted fish oil and hexanal) induced secondary structure changes in myosin over and above those caused by freezing. This was evidenced by decreased alpha-helix content with a concomitant increase of beta-sheet structure, indicating myosin polymerisation. A decrease in the tryptophan band, and increase in the ratio of the dityrosine bands indicated changes in hydrophobic groups. The model studies and the analysis of intact fish muscle of Gadus morhua and Melanogrammus aeglefinus suggest that protein denaturation occurred due to the concerted action of ice crystals, supercooled water molecules (unfrozen), high solute concentration, free fatty acids, and primary and secondary lipid oxidation products on the fish muscle proteins. Possible intervention schemes include, the addition of appropriate antifreeze glycoproteins, cryoprotectants and antioxidants.

Accurate and reliable measurement of meat quality is essential for the Australian beef industry to remain competitive in both the domestic and export markets. Recent developments of the resonator technique have lead to the commercial availability of the High Resolution Ultrasound Spectroscope (HR-US). This research project was designed to assess the potential of HR-US for the analysis of post-mortem bovine muscle. This was accomplished by; 1) establishing a suitable measurement protocol that considered sources of variability, 2) the effects of post-mortem aging on HR-US parameters, 3) analysis of thermal related changes that occur in muscle, and extracted connective tissue during heating, and 4) the use of HR-US for the measurement of the intramuscular fat. A procedure for the measurement of bovine muscle with HR-US was established. Briefly, an external semisolid cell was used as the measurement cell. The frequency range of 2000 KHz to 3000 KHz was selected as the most suitable for whole muscle analysis and all five resonance peaks within this range were analysed and used to obtain velocity and attenuation values of the meat sample. Water was used as the reference media, and measurements were conducted at 250C. Changes were made to this method during experimental work depending on the sample being run and the objectives of the study. The measurement protocol was shown to be repeatable. Factors likely to cause variation in measurements of the samples, such as water loss and freeze-thaw, were also considered when developing the operational parameters of the study. High resolution ultrasound spectroscopy was applied to measure the post-mortem changes that occur in bovine muscle. Using two muscle types, Semitendinosus and Psoas major, significant changes were observed in HR-US parameters with ageing. Significant increases in the acoustic impedance of bovine muscle with increased ageing time were attributed to degradation of the muscle structure. This was confirmed in transmission electron microscopy images where clear disruption the myofibillar structure was apparent in the muscle at 21 days post-mortem.In localised regions the Z bands and the adjoining actin fibres were totally degraded. Water loss from the muscle system had a significant influence on HR-US measurements. Thermal related changes that occur in whole bovine muscle and in isolated intramuscular connective tissue were observed with HR-US. Heat induced changes were identified in whole muscles and included the melting of the fat within samples at 48oC, coagulation of sarcoplasmic proteins between 450C and 55oC, and the shrinkage of collagen fibres at 630C. An 80% reduction in the attenuating properties of extracted connective ageing in buffer solution was observed within the first 5 days. This is attributed to the degradation of proteoglycans and the resulting disassociation of collagen fibrils. Structural changes occurring in extracted connective tissue were observed with TEM. HR-US measurements proved to be highly sensitive to identifying temperatures at which transitions occurred. Unfolding of the triple helix structure of collagen was identified in velocity transitions between 59°C and 63oC. HR-US results suggested a greater sensitivity to thermal related changes in extracted intramuscular connective tissue when compared with differential scanning calorimeter results. An increase in temperature was observed for thermal denaturation of collagen with ageing, however a reduction was also observed in the temperature range at which the denaturation process occurred. Temperature ramps conducted on extracted intramuscular bovine showed a reduction in velocity from 1613.1m/s at 250C to 1343.1 m/s at 900C equalling an overall reduction in velocity of 270m/s. A transition in the velocity trend seen at 46°C indicates the majority of the triglycerides are melted (or in liquid state) above this temperature. Results are confirmed with differential scanning calorimeter thermogram. HR-US measurements showed high sensitivity to increasing concentration of bovine fat in prepared emulsions with an adjusted R2 99.46% for velocity measurements taken at 5100 kHz. Attenuation values at 8100 kHz also showed a strong linear response to increasing fat concentration in the emulsion (R2 98.77). The use of HR-US for the measurement of intramuscular bovine fat demonstrated a high sensitivity to extracted bovine fat in prepared emulsions. An increase in the intramuscular fat content of whole bovine muscles resulted in a reduction in the velocity measurements and an increase in the attenuation of the ultrasonic signal. This provides the basis for potential method for the prediction intramuscular fat in bovine muscle. The present studies have highlighted the complexities of investigations relating to meat quality and have demonstrated the diversity of data required to assess quality. Only when comprehensive data are available, can we hope to accurately determine meat quality and predict how it will vary with changes in animal production and meat processing.

The objective of this project was to analyze the thermodynamics (thermal conductivity and diffusivity as well as protein denaturation) and physical properties (percent expressible moisture, cooking loss, change in steak thickness, shear force, texture profile analysis and rheological behavior) of beef steaks of different USDA quality grades (Upper 2/3 Choice and Select), thicknesses (thick and thin), and grill surface temperatures (high and low) cooked to the same internal degree of doneness to determine if a specific set of cooking parameters would create a profound difference in the eating characteristics, described by the tenderness and juiciness of cooked beef strip steaks. The elastic behavior of the surface and centers of beef steaks were analyzed to determine how the microstructure of the beef responded to applied stress. The elastic behavior of steak centers was influenced in a three-way interaction between USDA Quality Grade, steak thickness, and grill surface temperature while the elastic behavior of the surface of steaks was influenced only by USDA Quality Grade and steak thickness. These interactions along with the differences in the thermal characteristic of proteins suggest that the microstructure of beef steaks is significantly affected by each cooking treatment group. The physical properties in the beef steaks further support through more tangible applications that the composition, thickness, and cooking regiments impact the microstructure and thermal properties of beef and thus final tenderness and texture. This project identified cooking preparation should take into consideration that quality grade, thickness and cooking temperature will affect the textural eating qualities of beef steaks. Choice steaks were shown to be ideally sliced thick and cooked on a low grill surface temperature supported by the springiness, hardness, expressible moisture, and rheological data. Select steaks were not always effected by grill surface temperature and had similar results among the different measurements but the hardness, resilience and chewiness values along with viscosity suggest a thick steak cooked at a high grill surface temperature. Therefore, cooking parameters may be utilized as a mechanism to enhance beef steak palatability.

The possible existence of extraterrestrial life forms has been of interest to humans for many millennia. In the past few decades space travel has provided an opportunity to search life outside of Earth. Chiral molecules are critical molecules in Earth-based life and are among the first chemical molecules sought after as proof of potential extraterrestrial life; however, identification of these chiral molecules is difficult due the lack of sensitive instruments. The objective of this work is to develop a benchmark reaction to be used as a guide in the development of instrumentation, such as a polarimeter, to be used in the search for extraterrestrial life. To achieve this objective, to investigate the enzyme mandelate racemase (MR), which catalyzes the racemization between the enantiomers of mandelate. MR is a member of the enolase superfamily, which contains a (alpha/beta)7-b barrel domain, the fold most frequently found among all known protein structures. Activity of the enzyme was measured at low temperatures and in non-aqueous media, as these are the conditions that represent extraterrestrial terrain. We find that mandelate racemase (MR) is active in concentrated ammonium salt solutions and water-in-oil microemulsions in a temperature range between 30C to 70C; however, the enzyme is not active in several organic cryosolvents. The stability of the structure of MR was also explored. Using differential scanning calorimetry (DSC) we observe the unfolding of the enzyme was irreversible and therefore kinetically controlled. We also found proof for divergent evolution of the enolase superfamily, providing evidence for divergent evolution across the MR and muconate lactonizing enzyme (MLE) subfamilies has been demonstrated. However, we also conclude that reactions yielding a polarimetric signal, such as racemizations employed in this work, are suitable as a tool to find signs of life.

Thesis (M.S.)--Duquesne University, 2004. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 76-88) and abstract.

Abstract Beta-lactoglobulin (β-lg) is the main whey protein in bovine milk. It belongs to the lipocalin protein family, and it is one of the main milk allergens. Resistance to hydrolysis is a particular feature of β-lg making it possible that β-lg reaches the small intestine in its native form. Heat treatments during milk processing may change the native structure of bovine β-lg and change its intestinal transport properties. Heat induced conformational alterations may also expose new antigenic sites. However, there have been no previous studies on the effects of heat treatment on the transport of β-lg or on its sensitizing properties. Cow's milk allergy is one of the most important food allergies affecting about 2.4% of infants. Milk proteins, including β-lg, in breast milk substitute formulas are often the earliest foreign antigens in the diet of newborns. According to the hygiene hypothesis, natural infections and vaccinations may modify the immunological balance and decrease the risk of allergy. Isoelectric precipitations followed by anion exchange and gel filtration were used to purify bovine milk β-lg in its native form. Transport of native and heat-denatured β-lg was compared in two in vitro cell models, Caco-2 and M-cells. Sensitization properties of native and heat-denatured β-lg were studied with an animal model using Hooded-Lister rats. Effects of BCG vaccination in combination with the native β-lg were also studied. Effects of different sensitizations were assessed by antibody levels in serum and inflammation locally in the gastrointestinal tract. Heat denaturation of β-lg made its transport slower in both enterocytes and M-cells. M-cells were more effective transporters of both native and heat-denatured β-lg than caco-2 cells. Animals generated higher levels of IgE when sensitized with native β-lg, but heat-denatured β-lg induced a more intense inflammatory cell reaction in the gastrointestinal tract. Vaccination with BCG decreased serum IgE concentration and modified the predominant site of the inflammatory cell response in intestine. The results indicate that, heat denaturation of β-lg and BCG vaccination, change both the systemic and the mucosal response to bovine milk β-lg. The reasons for this remain speculative. The effect of BCG vaccination is consistent with the hygiene hypothesis. The observed alteration of transport properties could be one mechanism by which heat denaturation modifies the allergenic properties of this protein, but additional studies are necessary to assess whether other mechanisms, such as exposure of new antigenic determinants are also relevant.

yes / Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms.

No description available.

No description available.

Master of Science / Food Science Institute, Animal Science and Industry / Karen A. Schmidt / Yogurt is a good source of whey proteins, which have been reported to provide positive health benefits. During yogurt manufacture, the yogurt mix receives a heat treatment which pasteurizes the product, denatures the whey proteins affecting their availability, and enhances quality attributes. Thus the objective of this research was to improve the undenatured whey protein content in yogurt. The study was divided in two parts. The first part focused on the effect of pasteurization treatments of yogurt mixes (65 °C for 30 min vs. 90 °C for 10 min) on the yogurt firmness, G’, L*, syneresis and water holding capacity (WHC), and how these properties change as a function of storage. Nonfat dry milk (NFDM) was reconstituted (~11% w/v) pasteurized, cooled, inoculated with yogurt culture, incubated to pH 4.5, stored at 5 °C ±1 and evaluated for various physical and chemical properties on days 1, 15 and 29. The experiment was replicated 3 times and data were analyzed by SAS®. Yogurt samples had a 5-fold difference in whey protein denaturation (WPD) and the greater the WPD the greater the firmness, G’, L* and WHC but lesser the syneresis. During yogurt storage, L*, G’, syneresis and WHC increased. The second part of this research focused on whey protein concentrate (WPC) addition (3%) in yogurt mix combined with two pasteurization treatments (70 °C for 30 min vs. 90 °C for 10 min) to determine their effects on the yogurt quality. Yogurt mixes were formulated using 12.5% NFDM or 9.5% NFDM and 3% WPC and a procedure similar to the previous study was followed. The WPC addition resulted in a yogurt with decreased firmness, G’, WHC but increased syneresis. Yogurt made from mixes pasteurized at 90 °C for 10 min had ~60% WPD and comparable quality attributes regardless of WPC addition. Thus, additional WPC and less WPD in this study resulted in a yogurt with slightly lesser quality attributes but more undenatured whey proteins in the final yogurt.