There is a constant need of new synthetic emulsifiers in the food industry. Sugar esters are widely used as food grade synthetic emulsifiers, amongst which sucrose esters are the most common. Although sucrose esters are used very frequently, little is known about the use of lactose esters in food. There is a need for characterization of lactose esters before they can be used in foods. The objective of this study was to characterize a lactose ester, lactose monolaurate (LML) as an antimicrobial agent on food pathogens, evaluate its effect on 20 % oil-in-water emulsions as an emulsifier, and to explore its effect on crystallization behavior of anhydrous milk fat. In the first study (Chapter 3), the effect of LML was evaluated on survival of some Gram-positive and Gram-negative bacteria. For Listeria monocytogenes, a concentration of 1 mg/ml showed some inhibition in growth media whereas the cells were completely killed at 5 mg/ml. For Mycobacteria, an LML concentration between 0.1-1mg/ml was lethal. Scanning electron microscopy was also conducted to examine any changes in the morphology of cells. Listeria exhibited a change in morphology and a wrinkling effect was shown in Mycobacteria. In the second study (Chapter 4), the effect of LML as an emulsifier was evaluated in 20 % oil-in-water emulsions. The use level of LML was comparable to commercially available emulsifier polysorbate 20, and produced comparable stabilization in the emulsions upon use. In this study, an attempt was also made to optimize the synthesis of LML with respect to the immobilized enzyme and solvent combination. It was concluded that for 20 % oil-in-water emulsions, LML is a promising emulsifier at 0.5%. In the third study (Chapter 5), the effect of LML was evaluated at two concentrations on the crystallization behavior of anhydrous milk fat at two temperatures with high and low supercooling. On application of high intensity ultrasound (HIU) to anhydrous milk fat (AMF) at 31°C and 0.05 % LML the effect on viscosity of sample and crystallization behavior was evaluated. It was concluded that the viscosity of AMF decreased with the addition of 0.05% LML. The lower viscosity of anhydrous milk fat on addition of LML could be restored with the application of HIU.

Thèse de doctorat : Biologie cellulaire et moléculaire. Immunologie : Orléans : 2009. / Titre provenant de l'écran-titre.

Several pathogenic bacteria, including Listeria monocytogenes, use an F-actin-dependent motility process to spread between mammalian cells. Actin ‘comet tails’ propel Lm through the cytoplasm, resulting in bacteria-containing membrane protrusions that are internalized by neighboring cells. The mechanism by which L. monocytogenes overcomes cortical membrane tension to generate protrusions is unknown. In this work, I identify bacterial and host proteins that directly regulate the formation of protrusions. First, I show that efficient cell-cell spread in polarized epithelial cells requires the secreted Lm virulence protein, InlC. I next identify the mammalian adaptor protein Tuba as a ligand of InlC. InlC binds to a C-terminal SH3 domain in Tuba, which normally engages the human actin regulatory protein N-WASP. InlC promotes protrusion formation by inhibiting Tuba and N-WASP function, most likely by impairing binding of N-WASP to the Tuba SH3 domain. Tuba and N-WASP are known to control the structure of apical junctions in epithelial cells [1]. I demonstrate that, by inhibiting Tuba and N-WASP, InlC transforms taut apical cell-cell junctions into structures with a “slack” morphology. Experiments with Myosin II inhibitors indicate that InlC-mediated perturbation of cell junctions accounts for the role of this bacterial protein in protrusion formation. Collectively, my results suggest that InlC enhances bacterial dissemination by relieving cortical tension in apical junctions, thereby enhancing the ability of motile bacteria to deform the plasma membrane into protrusions to allow their spread into neighbouring cells.

Listeria monocytogenes has been found to withstand harsh environmental conditions including desiccation. The pathogen is also known to form biofilm when in co-culture with other bacteria found in food products. This study investigated the desiccation survival of L. monocytogenes in mixed biofilms with Pseudomonas fluorescens, Serratia liquefaciens and Shewanella putrefaciens. To this end, mono- or binary species biofilms were formed and desiccated (43% relative humidity, 21 days at 15°C) on stainless steel coupons and the double Weibull model was fitted to the resulting survivor curves. The presence of the competitor Gram-negative food spoilage bacteria with the exception of Sh. putrefaciens suppressed (p<0.05) L. monocytogenes during biofilm formation (100% relative humidity, 15°C and 48 h) and subsequently decreased (P<0.05) the desiccation survival in L. monocytogenes without affecting the resistance of individual cells. Microscopic approaches revealed different biofilm forming capabilities in the mono- and binary bacterial combinations.

The food processing industry requires alternative methods for detecting the foodborne pathogen Listeria monocytogenes that are cheaper and faster than the current methods. Conventional antibodies and their fragments have been used as biorecognition elements in sensors before, but their use is hindered by high production cost and relative instability. These issues are resolved by VHH fragments, derived from the heavy chain-only antibodies found in Camelidae. VHHs are inexpensive to produce, and are more resistant to environmental stressors. This work describes the isolation of phage-displayed VHHs that recognize recombinant Internalin B, a virulence factor characteristic of L. monocytogenes. Clone R303 was chosen for further characterization, and shown to bind full-length Internalin B. Furthermore, immobilized R303 was shown to capture L. monocytogenes cells. This panel of VHHs, particularly R303, can be utilized by colleagues within the Sentinel Bioactive Paper Network to make a viable biosensor for L. monocytogenes. / Sentinel Bioactive Paper Network

No description available.

A rapid detection method combining immunomagnetic separation (IMS), PCR and slot blot detection was developed for the detection of Salmonella and Listeria monocytogenes in milk. Bacteria were first isolated and concentrated from phosphate-buffered saline (PBS) or milk by IMS. After extraction from diluted bacteria culture with the extraction buffer, bacterial DNA was subjected to PCR. Slot blot assay was optimized and used to measure PCR products. The lowest level of detection by this method was 40 cfu/ml in PBS or milk for both pathogens. The whole detection procedure could be completed within 7 h. Moreover, this detection method is simple and easy to handle for a large number of samples. Using multiplex PCR (amplification of two different bacterial DNA in the same PCR tube) and slot blot, simultaneous detection of both bacteria was also assessed. The detection sensitivities of 103 cfu/ml for both bacteria were the same as when PCR and slot blot were used for each bacterium separately. The combination of IMS, PCR and slot blot seems to give a highly sensitive and time-efficient procedure, which could be used for routine detection of Salmonella and L. monocytogenes in milk.

Several pathogenic bacteria, including Listeria monocytogenes, use an F-actin-dependent motility process to spread between mammalian cells. Actin ‘comet tails’ propel Lm through the cytoplasm, resulting in bacteria-containing membrane protrusions that are internalized by neighboring cells. The mechanism by which L. monocytogenes overcomes cortical membrane tension to generate protrusions is unknown. In this work, I identify bacterial and host proteins that directly regulate the formation of protrusions. First, I show that efficient cell-cell spread in polarized epithelial cells requires the secreted Lm virulence protein, InlC. I next identify the mammalian adaptor protein Tuba as a ligand of InlC. InlC binds to a C-terminal SH3 domain in Tuba, which normally engages the human actin regulatory protein N-WASP. InlC promotes protrusion formation by inhibiting Tuba and N-WASP function, most likely by impairing binding of N-WASP to the Tuba SH3 domain. Tuba and N-WASP are known to control the structure of apical junctions in epithelial cells [1]. I demonstrate that, by inhibiting Tuba and N-WASP, InlC transforms taut apical cell-cell junctions into structures with a “slack” morphology. Experiments with Myosin II inhibitors indicate that InlC-mediated perturbation of cell junctions accounts for the role of this bacterial protein in protrusion formation. Collectively, my results suggest that InlC enhances bacterial dissemination by relieving cortical tension in apical junctions, thereby enhancing the ability of motile bacteria to deform the plasma membrane into protrusions to allow their spread into neighbouring cells.

Graduation date: 1993

Errata and corrections pasted on front end paper. / Copies of three of author's previously published articles contained in back pocket. / Bibliography: leaves 178-219. / ix, 219, [130] leaves, [31] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis aims to generate a library of chromosomally derived transcriptional promoter ::lacZ fusion mutants in an environmental isolate of L. monocytogenes (DRDC8). A lacZ transcriptional fusion mutant that displayed increased B-galactosidase activity in response to the calcium chelater EGTA is investigated in detail. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1997