by Poon Hoi To. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1981. / Bibliography: leaves 40-41.

Clinical thermal therapies are widespread and gaining in appeal due to improved technology of heating devices and promising results. Outcomes of thermal treatment are often unpredictable and suboptimal, however, due in part to a lack of appreciation of the underlying biothermomechanics. There is a pressing need, therefore, to understand better the role of clinically-controllable parameters on the thermal damage processes of tissue. Heretofore, researchers have primarily sought to understand this process through various uniaxial experiments on tissues containing collagen as their primary constituent. Most biological tissues experience multiaxial loading, however, with complex boundary constraints inclusive of both isotonic and isometric conditions. The primary focus of this work is on the isothermal denaturation of fibrillar collagen subjected to a biaxial isometric constraint. Results from our tests reveal a complicated process, the kinetics of which are not easily measured. Evolving isometric contraction forces during heating do not correlate with resultant mechanical behaviors, as thermal shrinkage does in biaxial isotonic tests. Furthermore, resultant mechanical behaviors at variousdurations of heating reveal a two phase process with a rate dependent on the amount of isometric stretch. For tissues heated at 75oC for 15 minutes, at which point the first phase of mechanical alteration dominates for all constraints herein, resultant mechanical behaviors correlate well with the amount of isometric stretch. The correlation is similar to that between isotonic loads and resultant mechanical behaviors from previous studies. In light of the need for a better measure of thermal damage in isometric tests, we performed a histological analysis of tissues heated under varying constraints. Results show a good correlation between the level of isometric constraint and thermally-induced histological aberrations. Finally, we demonstrate that our seemingly limited and qualitative knowledge can be applied well to a specific clinical application: namely, the use of glycerol as a clearing agent for laser therapies. Our results suggest that glycerol is safe to use for such therapies because it increases the thermal stability of fibrillar collagen, and its hyperosmotic effects on mechanical behavior are fully reversed upon rehydration.

Myofibrillar proteins, the main components that impart functional properties to muscle foods, can undergo denaturation and aggregation during frozen storage. The overall objective of this research was to study the changes in protein structure that are associated with the preparation and frozen storage of surimi. In addition, the relative cryoprotective effects of whey protein concentrate, whey protein isolate, soy protein isolate, flaxseed meal and flaxseed protein were assessed in surimi during storage. / Raw surimi was prepared by repeatedly washing Alaska pollock flesh with chilled water. The product was either slowly frozen or underwent rapid freezing using liquid air; in either case it was then subjected to frozen storage at -20°C for 24 months. Protein structural changes were monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), native-PAGE, Fourier transform infrared/attenuated total reflectance (FTIR/ATR) spectroscopy, and differential scanning calorimetry (DSC). / FTIR/ATR spectroscopy showed that during preparation of surimi the alpha-helix content increased with increased number of washing cycles. DSC results revealed a shift in the thermal transition of actin to a higher temperature during surimi preparation. All electrophoresis, FTIR/ATR spectroscopy and DSC results revealed a loss of myofibrillar proteins from surimi after three washing cycles, suggesting that three washing cycles were adequate to prepare surimi. / Native-PAGE showed no major changes in surimi after 24 months storage at -20°C. SDS-PAGE showed relatively minor changes in protein subunit structure with some loss of the myosin light chains (MLC); myosin heavy chain (MHC), actin and tropomyosin were found to be relatively stable. FTIR/ATR spectroscopy indicated a significant decrease in alpha-helix relative to beta-sheet structure in surimi after 2 years of storage at -20°C. The loss of alpha-helical content was more significant in slowly frozen surimi compared to rapid-frozen surimi samples. DSC results revealed a shift in the thermal transition of actin to lower temperatures during frozen storage of surimi. / Changes in the ratio of alpha-helix to beta-sheet structures suggested that flaxseed protein was the most effective cryoprotectant, followed by whey protein isolate and soy protein isolate, for maintaining protein structure stability during frozen storage. Whey protein concentrate and flaxseed meal showed the least cryoprotective ability. After 15 days storage at 4°C, the SDS-PAGE results showed that flaxseed protein was the only cryoprotectant that prevented the degradation of myosin heavy chain, actin and myosin light chains.

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.

No description available.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

A casein agar diffusion method was developed to detect and quantify pertinent levels of proteinases produced in raw milk supplies by heat resistant Bacillus sporeformers. In order to optimize the required heat treatment conditions of raw milk samples, trials that involved a combination of different temperatures and times were evaluated. A heat treatment of 75°C for 20 min was the most effective for recovering the highest number of surviving spores. A sporulation broth containing five different minerals and supplemented with 0.2% nonfat dry milk was used to maximize spore production in all heat-treated samples. A β-casein based assay detected proteinase activity from raw milk samples that ranged from 0.093 to 4.034 units/mg which corresponded to zones of β-casein precipitation in the β-casein agar of 5.0 and 15.0 mm respectively, and was compared to Protease Type VIII (from B. licheniformis). This assay correlated well with the fluorescein isothiocyanate casein-labeled assay (FITC), R=0.995 (Protease Type VIII). Proteases of Bacillus origin such as Protease Type IX, X, XV and XXXI were also evaluated but were rejected in favor of a broader range of activity expressed by Protease Type VIII. For an initial set of 370 raw milk samples, no quality deterioration, such as coagulation or bitter taste was observed in heat-treated (75°C for 20 min) and incubated samples (7.2°C for 10 days). However, during the winter season, 18 of 75 incubated samples (7.2°C for 10 days) tasted slightly bitter and exhibited a slight degree of casein precipitation. One sample coagulated but exhibited no proteinase activity on the β-casein agar gel, hence it was considered a false negative. The positive results for proteinase activity from raw Grade A samples tested by the β-casein agar diffusion method did not correlate either with fresh spore counts (R=0.21) or post-heat treatment incubation counts (R=0.03) or with psychrotrophic sporeformer counts (R=0.06). The β-casein agar diffusion method is simple, rapid and sensitive to Bacillus spp. proteinases, but was unreliable in projecting results related to the psychrotrophic sporeformer count. Consequently, further research is required to establish optimum conditions (time and/or temperature) and inoculum volumes into sporulation broth for attainment of a more positive correlation between β-casein agar precipitation zones and psychrotrophic sporeformer populations of either raw or processed milk samples. / Graduation date: 1991

Guanidine-stable chymoelastase (GUSCE), a component of the protease mixture known as Pronase, has been shown to be stable and active under conditions which denature and inactivate most proteins. In the absence of denaturant, this enzyme has shown proteolytic specificity for phenylalanyl, tyrosyl, and leucyl peptide bonds. In the presence of denaturant, however, the cleavage specificity has not been defined.In order to determine the effects of denaturant on the cleavage specificity of GUSCE, six small peptides of known amino acid sequence were hydrolyzed by GUSCE in the presence and absence of 6.OM guanidinium chloride. The site of GUSCE cleavage was acertained by dansylation of the new N-terminal amino acids, which were produced by proteolysis, followed by thin layer chromatographic identification of the resulting dansylated amino acids.The results indicate that GUSCE catalyzed the hydrolysis of phenylalanyl and tyrosyl peptide bonds in the absence as well as the presence of 6.OM guanidinium chloride. Of the tyrosyl and phenylalanyl peptide bonds hydrolyzed, all were between non-terminal amino acids, which illistrates the endo-peptidase characteristics of GUSCE. With one exception, only those peptide bonds cleaved by GUSCE in the absence of denaturant were cleaved in the presence of denaturant. In the case of oxytocin, the presence of denaturant was actually required for the cleavage of the Tyr(2)-Ile(3) peptide bond. The demonstrated predictablilty of GUSCE cleavage in the presence of denaturant should greatly enhance its utility in the sitespecific proteolysis of insoluble or otherwise proteolysisresistant protein substrates.Ball State UniversityMuncie, IN 47306

Graduation date: 1993

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 181-216) Also available in print.