The application of infrared imaging employing focal plane array Fourier transform infrared (FPA-FTIR) instrumentation for the identification of bacteria was investigated. FPA-FTIR spectroscopy was shown to provide new opportunities for bacteria identification with unprecedented reliability and throughput by allowing 102--103 FTIR spectra to be acquired simultaneously from surface areas of 90 x 90 to 200 x 200 mum with a spatial resolution of ∼6 mum. The combination of data redundancy and spatial resolution afforded by infrared imaging made it possible to acquire highly reproducible spectra from bacterial films. A protocol for enhancing the reliability of bacteria identification by transmission-mode FPA-FTIR spectroscopy was developed by optimizing spectral acquisition parameters, spectral processing and data analysis; using the differentiation of two Campylobacter species as a test case. The results for this test case were compared with those obtained from three alternate FTIR spectral acquisition modes. The optimized protocol was employed for the generation of a spectral database of foodborne bacteria, containing over 1,000,000 spectra acquired by infrared imaging of 36 species from 19 genera. The development of a modular hierarchical clustering (MHC) model, in combination with the use of a region selection algorithm, allowed all species in the database to be differentiated from each other down to the species level based on differences in their infrared absorption profiles. A validation study involving the identification of well-characterized isolates by comparison of their spectra to those in the database demonstrated the robustness of the MHC model. In a further study employing 44 strains of Clostridium botulinum, the discriminatory power of FPA-FTIR spectroscopy was compared with that of pulsed-field gel electrophoresis, and the region selection algorithm was applied to identify growth medium-independent spectral regions that allowed for the differentiation of Group I and Group II C. botulinum strains in two blind validation studies. The research carried out also demonstrated the high-throughput potential of bacteria identification by infrared imaging when combined with the use of a microarray system for sample deposition. Overall, the novel FPA-FTIR spectroscopy-based bacteria identification protocol developed in this work provides a rapid-response and reagent-free technique suitable for routine use in both food and clinical microbiology laboratories.

Globular proteins are important ingredients in many food formulations because of their nutritional value and their various functional properties such as gel formation. Proteins form gels by polymerization into a three-dimensional matrix. The rheological properties of the resulting viscoelastic solids can be obtained by Instron measurements. In the present work, gels were obtained by heating solutions of bovine serum albumin (BSA) in D$ sb2$O and egg albumin in H$ sb2$O under different conditions (i.e., pH, salt concentration, protein concentration, time of heating and temperature), and their gel strengths were measured by a compression test (Universal Testing Machine LRX). The Fourier transform infrared (FTIR) spectra of the same gel samples were recorded in order to investigate the changes in protein structure at the molecular level accompanying gel formation. Intermolecular $ beta$-sheet formation was found to increase as gel formation progresses at the expense of intramolecular $ beta$-sheet and $ alpha$-helix structures. For BSA, maximum gel strength was obtained around pH 7. The addition of salt had little effect on the gel strength while increase in protein concentration, time of heating and temperature increased the gel strength. The rate of denaturation of BSA and ovalbumin and of mixtures of these proteins in the ratios 9:1, 1:1, and 1:9 was also investigated by measuring the peak height of an aggregation band at 1618 cm$ sp{-1}$; some inhibitory effects of BSA on ovalbumin aggregation were observed. Correlations between the measured gel strengths and the amide I band profile in the FTIR spectra were examined using partial-least-squares (PLS) regression. These studies reveal that gel strengths of a particular protein gel could be adequately predicted from their infrared spectra without the need to carry out the rheological compression measurements.

This work describes an investigation of the development of a Fourier Transform Infrared (FTIR) spectroscopic method for the determination of Solid Fat Index (SFI) of fats as a possible replacement of the traditional dilatometric procedure. The initial approach considered was the use of an existing FTIR analytical package which was designed to measure iodine value (IV), saponification number (SN), and cis and trans content. It was hypothesized that these measures could be related to SFI using multiple linear regression (MLR), thereby allowing the existing analytical package to simultaneously make SFI measures. It was found that there was a strong relationship between SN/cis/trans measurements and SFI, especially in sequentially hydrogenated oils. The MLR relationships, however, did not reproduce the dilatometric SFI values with sufficient accuracy in the general case, and this approach had to be abandoned. Subsequently, a partial least squares (PLS) calibration approach was investigated, relating the dilatometric SFI data directly to the spectral characteristics of the melted fats. It was found that suitable PLS calibrations could be developed for soybean and Canola oils. Based on these results, an FTIR system was programmed to determine SFI and the performance of the system validated using pre-analyzed "unknowns". It was shown that the SFI of either soybean or Canola oils could be determined to within $ pm$ 1.0 SFI. As configured, the FTIR system is capable of determining the SFI of a neat and clear, melted fat sample at 80$ sp circ$C in less than two minutes, providing four SFI values, representing the solids content at 50, 70, 80 and 92$ sp circ$F. In contrast to the standard dilatometric method, which takes over two hours to carry out, the FTIR approach provides a rapid means of determining SFI, the technique being suitable for routine quality control applications in the fats and oils industry.

Staphylococci strains are among the most widespread multidrug-resistant nosocomial pathogens in Canada. Rapid and accurate identification and epidemiological typing of methicillin-resistant S. aureus (MRSA) and its discrimination from coagulase-negative staphylococci (CNS) and glycopeptide-intermediate S. aureus (GISA) are crucial for appropriate therapy and for monitoring and limiting intra- and inter-hospital spread of epidemic MRSA strains. Although pulsed-field gel electrophoresis and polymerase chain reaction methods for the identification of MRSA are reliable, they are technically demanding, time-consuming and inappropriate for routine clinical diagnosis. Moreover, no reliable method exists for discrimination of epidemic MRSA from sporadic MRSA and from GISA strains. The objective of the research described in this thesis was to investigate whether Fourier transform infrared (FTIR) spectroscopy could be used to distinguish MRSA from methicillin-susceptible S. aureus, borderline oxacillin-resistant S. aureus (BORSA), CNS, including methicillin-resistant CNS, and GISA. The application of FTIR spectroscopy for epidemiological typing of Canadian epidemic MRSA (CMRSA) strains as well as their discrimination from sporadic MRSA was also assessed. FTIR spectra were recorded from intact stationary-phase cells grown on Universal Medium (UM™) and deposited and dried on a ZnSe optical window, normalized, and converted to first-derivative spectra. Various chemometric approaches were employed to cluster the different phenotypes of staphylococci species and to subtype five CMRSA strains based on the similarity of their infrared spectral fingerprints in narrow spectral regions selected by visual inspection and by employing a singularvalue decomposition (SVD) algorithm. Pairwise separation of MRSA from MSSA, BORSA, CNS, MRCNS, and GISA was accomplished by using principal component analysis (PCA), self-organizing maps (SOM), and the K-nearest neighbors (KNN) algorithm. These chemometric techniques were also successfully employed for epidemiological typing of the five CMRSA strains and their discrimination from sporadic MRSA strains using a combination of different optimal spectral regions selected by SVD. These results demonstrate that FTIR spectroscopy has considerable potential as a rapid method for the identification of different phenotypes of staphylococci and epidemiological typing of MRSA.

Variable-temperature Fourier transform infrared spectroscopy can be employed to monitor the denaturation and aggregation of proteins during heat treatment. Information on the changes that occur in protein secondary structure upon heating is provided by detailed examination of the amide I band, as different protein conformations have characteristic amide I frequencies. The objectives of the present study were: (i) to study the changes in protein structure that occur during gelation of whey protein isolate (WPI) and (ii) to correlate the changes in protein structure observed under different physico-chemical conditions to rheological properties of WPI gels prepared under the same conditions. / The FTIR spectra of D2O solutions of WPI at different pHs, ranging from 3 to 10, were recorded as the temperature of the solution was increased from room temperature to 95°C in 5°C increments. In all the solutions studied, the formation of two new bands at 1618--1623 cm -1 and 1678--1684 cm-1 was observed upon heating; these bands are characteristic of aggregate formation and have been previously assigned to antiparallel beta-sheet structure. As the pH increased from 3 to 10, the aggregation temperature of WPI decreased from 85°C to 65°C. / The rheological properties of WPI gels were studied by employing an Instron Universal Testing Machine. The Instron measurements demonstrated that protein concentration, heating temperature, and heating and cooling time are directly related to gel strength. The changes in gel strength as a function of cooling time (for gels prepared by heating at 75°C for 45 min) were correlated to FTIR spectral data for WPI solutions subjected to the same treatment.

Recent studies have demonstrated that applied hydrostatic pressure can affect the functional properties of whey protein isolate (WPI). In this work, the effects of applied hydrostatic pressure on the tertiary and secondary structure of whey proteins were investigated by spectroscopic and rheological techniques to elucidate the molecular basis of such pressure-induced changes in protein functionality. The individual protein components of WPI and various samples of WPI obtained from different sources were subjected to different single-cycle pressure treatments of up to 400 MPa in 100 MPa increments with 30-min holding time as well as to pressures ranging from 450 to 650 MPa without a holding time. Electrospray ionization-mass spectrometry, circular dichroism, and Fourier transform Raman spectroscopic studies of pressure-treated samples of beta-lactoglobulin, the major protein component of WPI, revealed significant changes in tertiary structure. Fourier transform infrared spectroscopic studies revealed that the secondary structure of beta-lactoglobulin was also sensitive to applied pressure and holding time. The secondary and tertiary structure of alpha-lactalbumin, the second most prevalent protein in WPI, was unaffected by applied hydrostatic pressure. The spectroscopic behaviour of the various samples of WPI subjected to pressure treatment was variable and indicated that the response of WPI to applied hydrostatic pressure is dependent on the method used to isolate the WPI from whey. The rheological profiles of beta-lactoglobulin, alpha-lactalbumin, and WPI samples after various pressure treatments were also recorded. Both beta-lactoglobulin and WPI exhibited marked increases in viscosity with increasing pressure, whereas alpha-lactalbumin remained solutions exhibited no significant change in viscosity. These studies have furthered the understanding of the effects of applied hydrostatic pressure on the molecular structure and rheological pr

Fourier transform infrared (FTIR) spectroscopy is a powerful tool for probing protein structure-function relationships. With the use of isotope editing, it can also be employed to elucidate protein-nucleic acid interactions. This technique was used to study the sequence of heat-induced unfolding of the uniformly labeled 13C regulatory subunit (RSU) of E. coli aspartate transcarbamylase (ATCase) with its inhibitor CTP. The absorption of CTP in the amide I' region limits our ability to detect protein conformational changes upon binding of CTP. Therefore, by labeling the protein with 13C shifts the amide I' band ~ 40 cm -1 and clearly separates the protein bands from those of CTP. Variable-temperature (VT) FTIR spectroscopy was then employed to monitor the thermal unfolding of the labeled RSU in the presence and absence of CTP. / In addition, isotope editing was further explored to probe domain-domain interactions of the two domains of RSU using intein technology. Intein technology provides a novel means by which isotope editing can be performed to extract information on protein inter-domain and inter-subunit interactions by spectroscopic analysis but has not yet been exploited in Fourier transform infrared (FTIR) spectroscopy. The objective of this project is to present for the first time the feasibility of segmental labeling through intein-mediated protein ligation (IPL) for the purpose of studying conformational changes by FTIR spectroscopy, using ATCase as a model enzyme. In the first phase of this project, the RSU of ATCase, which houses a Zn-binding domain and a nucleotide binding domain, was reconstructed from its isolated domains using commercially available intein-base expression vectors. As steps towards obtaining an isotope labeled RSU, we have fused each domain to separate inteins. Following affinity purification, the intein tags were chemically cleaved and the reactive ends of the two RSU domains were ligated together to form a peptide. Although ligation was successful, improved yields are required for the FTIR spectroscopic studies.

Defatted soybean was subjected to isolation procedures as follows: NaOH extraction/acid precipitation (NaOH-IE); water extraction/acid precipitation (H2O-IE); NaOH extraction/cryoprecipitation (NaOH-CP) and citric acid extraction/cryoprecipitation (CA-CP). NaOH-CP and CA-CP each resulted in precipitation of one protein mainly consisting of glycinin and beta-conglycinin, respectively. The proteins were subjected to polyacrylamide gel electrophoresis (PAGE), SDS-PAGE, size exclusion-chromatography (SEC) and differential scanning calorimetry (DSC) for characterization. PAGE, SDS-PAGE, SEC and DSC confirmed the homogeneity of NaOH-CP. The gelation properties and structural characteristics of NaOH-CP were investigated; the minimum protein concentration required for formation of a thermally-induced gel from NaOH-CP was 8%. With cold-set gelation, in the presence of 30 mM CaCl2, the minimum protein concentration required for formation of a self-supporting gel was 4%. At 100 mM CaCl 2, cold-set gelation of 10% protein concentration gave gel of strength 7.4 N compared to the 7.8 N gel strength obtained with thermally-induced gel at 16% protein concentration. The effects of heating on the NaOH-CP structure, in the presence of varying concentrations of NaCl and reducing agents, were examined by Fourier transform infrared (FTIR) spectroscopy and DSC. FTIR spectroscopy revealed that at 25°C the NaOH-CP protein showed four bands at 1633, 1650, 1667, and 1689 cm-1. At 85°C, the temperature associated with gelation, the four bands decreased in intensity with the simultaneous increase in the intensity of a band at 1644 cm-1 as result of the protein unfolding, furthermore two new bands at 1616 and 1685 cm -1 appeared. The latter two bands are attributed to intermolecular mu-sheet formed during gelation. The NaOH-CP did not exhibit any aggregation band between 25--100°C in the presence of NaCl. The DSC studies revealed that the protein denatured at approximately 102°

Polymeric depressant adsorption on the surface of talc was investigated using in situ Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy. The polymeric depressants investigated were an unmodified polyacrylamide (Polymer-N) and a hydroxyl substituted polyacrylamide (Polymer-H), an unmodified dextrin (Dextrin WY) and a hydroxylpropyl substituted dextrin (HP Dextrin), and carboxymethyl cellulose (CMC). The main objectives of this study were to evaluate the thermodynamic properties of adsorption, the kinetics of adsorption and desorption, and the mechanisms driving adsorption. / Thesis (PhDApSc(MineralsandMaterials))--University of South Australia, 2007.

Thin coatings of photoresponsive, pyrimidine-terminated molecules, attached to gold or quartz substrates in contact with water, undego photodimerisation and wettability changes when irradiated with UV light at 280 and 240mm. Spin-casting and chemisorption techniques were used to prepare the thin films. / Thesis (PhDApSc)--University of South Australia, 2000.