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    Shear stress adaptation of Listeria monocytogenes in mono and dual-species biofilms
    (Elsevier Ltd, 2025-12-01) Pant K; Palmer J; Flint S
    While the impact of stress on L. monocytogenes associated with food processing has been recognized in planktonic conditions, the available research overlooks the response of this pathogen in the multi-species biofilm, commonly found in food processing and manufacture. The objective of this study was to understand the effect of shear stress on L. monocytogenes in single and dual-species (with P. fluorescens) biofilm formed in a continuous turbulent flow system. In the single-species biofilm, L. monocytogenes was able to form a biofilm under the turbulent flow with cell concentration reaching 5.1 log CFU/cm2 after 48 h, where filamentous cells (27.7 μm in length) were observed. In contrast, there were no visible filaments in the dual-species biofilm, and L. monocytogenes cell concentration was significantly higher (p < 0.001) at 8.7 log CFU/cm2. The cells harvested from single-species L. monocytogenes biofilm formed under turbulent flow showed significantly (p < 0.001) lower motility and higher adhesion compared with cells harvested from planktonic and static conditions. Gene expression analysis showed significant (p < 0.001) downregulation of motB (motility), sigB (stress), and cell division (ftsX and ftsW), and upregulation of mpl (adhesion) and rodA (rod shape), indicating L. monocytogenes adaptation to shear stress. This study provides fundamental information on the multi-species biofilm formation by L. monocytogenes under stress.
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    Adherence interactions between milk proteins and human intestinal surface layer components : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, Palmerston North, New Zealand
    (Massey University, 2016) Schmidmeier, Christiane
    Recent research suggests a number of food-derived proteins may be used as orally delivered functional components. The native structure is often vital to their activity and requires protection during the digestive process. Nutrient vehicles are used as protective envelopes and as a mechanism for targeting specific sites of activity, e.g. the small intestine. This study evaluated molecules which adhere to one or more in vitro models of three human intestinal surface layers. Successful candidates could then be incorporated into nutrient vehicles, promoting adhesion to the surface layers and resulting in prolonged retention of the active ingredient at the site of action or absorption. To identify molecules that adhere to models of the intestinal surface, an adhesion protocol was developed to screen the proteome of whole milk, skim milk and whey for candidate proteins. Molecules adhering to model layers of the human gastrointestinal tract (intestinal epithelial cells, mucin or bacteria with the propensity to form a biofilm) were screened by SDS-PAGE analysis and identified by mass spectrometry and Western blot. The binding behaviour of selected proteins was further investigated by flow cytometry. The combined results showed that milk and whey proteins exhibit different binding affinities to the models of individual surface layers. a-Lactalbumin was found to adhere to a model of the intestinal epithelial cells, while ß-lactoglobulin showed binding to the protective mucin layer. Lactoferrin and various components of immunoglobulins showed highest binding affinity to bacteria. Finally, IgM appeared to adhere to all three tested model layers of the human gastrointestinal surface. Least binding was observed to the intestinal epithelial cells in culture. The validity of the developed adhesion protocol was demonstrated by replicating adhesion of immune-related proteins, lactoferrin and immunoglobulins, to bacterial cells. This work reveals new important characteristics of milk-derived proteins in their ability to adhere to models of the gastrointestinal surface. These may be further utilised in site-specific targeting of functional foods.
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    The adhesion force study of dairy thermophile Anoxybacillus flavithermus CM with atomic force microscopy : a thesis presented in partial fulfilment of the requirements for the degree of Master of Engineering in Chemical & Nanotechnology at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Mohd Saidi, Mohd Salihin
    Anoxybacillus flavithermus is a common species of thermophilic bacteria discovered in most milk powder manufacturing plants through out New Zealand. The contamination of it’s spores into the finished milk powder is an on-going problem as these spores are able to survive the sterilization process. Cheating death, A. flavithermus spores were then believed to attached on the stainless steel surface piping of the production line and germinate into a mature bacteria. A single surviving spore could grow to produce more spores that eventually dislodged from the colony and deposited together with the packaged milk powder. Over the storage time, the contaminated product will gives an off flavor as it deteriorates from bacterial action within. Currently, the applied cleaning method is by rinsing the target section with 1% sodium hydroxide & acid solutions before being flushed out to remove any microorganisms attached on the interior surfaces. However, it is not very effective in removing spores and there is very little information on the value of the spore’s adhesion force on a stainless steel surface. With that in mind, the aim of this study is to determine a proper adhesion force value between a dairy strain spore, A.flavithermus CM and stainless steel surface using the Atomic Force Microscopy (AFM) system. Meanwhile, Geobacillus strearothermophilus ATCC 2641 which is also a thermophilic organism was used over the study for comparison purpose. To measure the adhesion force under an Atomic Force Microscopy (AFM), the crude suspension was first purified using two-phase separation method. Polyethylene glycol (PEG) and phosphate buffer were used as the phase separation chemicals while 0.1% polysorbate 20 was added to the freshly purified spores’ suspension to aid the imaging sequence under the AFM. All AFM imaging and force measurements were done in air and conducted using the silicon type CSG 11/Au cantilever. The crucial Force-Volume imaging was done on a 32x32 grid scan size (1024 samples) on a scan rate of 0.5 Hz. It was calculated that a single A. flavithermus CM spore has an adhesive force value of 16.8 µN when attached on a stainless steel surface. It has a stronger localize adhesive value of 3.9 nN than a G.stearothermophilus ATCC 2641 spore with just 3.6 nN. However, G.stearothermophilus ATCC 2641 has a larger adhesive force of 21.1 µN on a stainless steel surface due to it’s larger spore size. It was also found that spore’s hydrophobicity does not dictates the magnitude of it’s adhesion on any surface. The results from this study have provide the dairy industry an extra sight on the quantitative value of the adhesion force of thermophilic spores, particularly A.flavithermus CM. This will help the dairy industry to design strategies in preventing spores from adhering to its production lines.
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    Bacterial attachment to meat surfaces : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2003) Narendran, Valarmathi
    The aim of this study was to optimise the hygienic efficiency of slaughter and dressing operations. Three strategic approaches, namely reducing and removing or killing the bacteria attached to meat surfaces, were considered. The second option of removal was selected for development, as current technology inevitably results in bacterial contamination, while killing bacteria on meat surfaces requires drastic treatments that may adversely affect quality parameters. The initial attachment mechanism between bacteria and the carcass surface (reversible attachment) was studied using the collagen film model system. Bacterial attachment to the collagen model was compared with attachment to cut beef muscle and uncut beef muscle using viable count procedure. Scanning electron microscopy and direct microscopic count procedure using an epifluorescence microscope was also developed using both collagen films mounted on microscope slides and collagen coated microscope slides. The collagen film viable count system was the method selected to model bacterial attachment to meat because of ease and consistency of quantification. There was no positive correlation between attachment and many bacterial cell surface factors such as charge, hydrophobicity, protein and polysaccharide surface molecules. Different eluents were used to identify the principal component interfering with single attachment mechanisms on electrostatic interaction and hydrophobic interaction chromatographic columns and on collagen film. Three components interfering with the isolated attachment mechanisms were identified. They were Tween, sodium chloride (NaCl) and mannose. Further column studies indicated that cell surface proteins play a more important role in cell surface negative charge and hydrophobicity than do surface polysaccharides. A wash solution was formulated using the components Tween, NaCl and mannose to reverse what were believed to be the major attachment mechanisms. Further trials with Tween, NaCl and mannose and increasing their concentrations and the application of increased vigorous rinsing also proved ineffective for washing the cells from meat surfaces. This result also supports the hypothesis that bacterial attachment to meat surface is very complex and multifactorial. Elution studies using 10 % Tri sodium orthophosphate pH 12.0 killed the cells rather than removing them and further work will be directed towards the killing.