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The production of olive oil has seen an increase in recent years due to a broader understanding of the health benefits of the Mediterranean Aliment Culture. With this expanding industry we also see an increase in the waste products associated with olive oil production. Given the high polluting content of the waste streams and the economic costs associated with its removal and processing, waste remediation and disposal has become a significant point of interest for both producers and local bodies. In this project, wastes of the olive oil production industry are examined for their use as the raw material for a novel product used in the control of horticulturally important diseases, examining the effect of extraction protocols on the activity of the final product. Active fractions of the olive oil wastes were identified from literature and protocols for their extraction and recovery developed; incorporating both standard solvent extraction and novel ultrasound-assisted extraction. Criteria for the analysis of extract quality were outlined and potential target applications identified. The biophenolic compounds of olive wastes were identified as providing the majority of the active fraction, so protocols were developed for the recovery of these compounds. Standard solvent extraction and ultrasound-assisted extraction were examined for their effectiveness of biophenolic recovery and their effect on product quality. Certain horticulturally important diseases were identified as potential targets, and bioassays undertaken to determine the ability of a crude extract to inhibit and control these diseases. It was found that the action of ultrasound during extraction provides a greater degree of recovery of biophenolic compounds, with minimal loss of product quality; as determined by bioassays and total biophenol determination. This increase in recovery is due primarily to the destruction of cellular material resulting in higher rates and absolute yields of recovery. This work provides evidence of the occurrence of some interesting phenomenon in the recovery of biophenols from olive wastes that deserves further examination. The crude olive leaf extract was shown to have an inhibitory effect on bacteria and effectively no inhibitory effect on fungal species in the total biophenol ranges tested. Erwinia amylovora and Staphylococcus aureus both showed a large susceptibility to the olive leaf extract. Results showed a higher degree of susceptibility of Gram positive bacteria and a potential resistance in soil microbes. For bacterial species, total biophenol concentrations of 0.15 to 3.50 mg GAE/ml provided inhibitory effects, while with the fungal species tested, no inhibitory effects were found at total biophenol concentrations of up to 2.50 mg GAE/ml. Some evidence exists that there is an opportunity for the economic recovery of olive biophenols for use as a novel product, but more work is required to determine specific applications and/or targets of use, as well as optimisation of the extraction and purification protocol. A sample removed from interfering compounds will allow the examination of activity of particular compounds and hence a better understanding of the action of the olive waste extract.

The aim of this study was to observe biofilm formation by Enterobacter sakazakii (E. Sakazakii) from different clinical, dairy and environmental origins on three infant feeding tubes made of different materials. Infant formula milk was selected as the medium for E. sakazakii growth. Seventeen isolates from different origins were retrieved and tested for purity, using a plating method and biochemical tests to eliminate the non E. sakazakii strains from this study. A method to rapidly and accurately detect viable cells of E. sakazakii on infant feeding tube surfaces using of the BacTrac® 4000 microbiological growth analyser was developed. The sources of errors such as from cleaning, operation and handling procedures were assessed prior to experimental runs. The strength of biofilm formation by different isolates of E. sakazakii on plastic surfaces was scrutinised using a microtiter plate assay. The results from the microtitre plate assay were based on the absorbance at 550 nm of crystal violet stained films and showed that all the clinical isolates were able to attach and form strong biofilms on the plate. Some environmental isolates formed strong or weak biofilms and some did not produce biofilm at all. However, dairy isolates formed both strong and weak biofilms in the microtitre plate when incubated in 10% reconstituted infant formula milk. The further studies were to quantify biofilm formation by three isolates of different origin on three different materials of infant feeding tubes using a batch system. Tubing pieces were incubated with infant formula milk inoculated with E. sakazakii cells at approximately 8 log CFU mL-1 and the biofilm formation was assessed at three time intervals: 4, 12 and 24 hours. Biofilm formation on the tubing by clinical isolates was also observed using epifluorescence microscopy and the scanning electron microscope. E. sakazakii from clinical, dairy and environmental isolates were able to form biofilm on three different materials of infant feeding tubes. The results showed that the initial attachment at 4 h on silicone tubing was low compared with the other two tubes. The scanning electron micrographs showed the surface characteristics of each tubing and the biofilm formation by E. sakazakii clinical isolates after 4, 12 and 24 hours. Silicone tubing appeared to be the best choice for premature babies that need feeding using feeding tubes, as it was slow to become colonised compared with the PVC and polyurethane tubing.

Polyhydroxyalkanoates (PHAs) are polyesters composed of (R)-3-hydroxy-fatty acids. A variety of gram-positive as well as gram-negative bacteria and some archaea are able to produce these biopolymers as energy and carbon storage materials. In times of unbalanced growth, when carbon is available in excess but other nutrients are limited, PHA inclusions are formed. These granules are water-insoluble, stored intracellularly and can be maintained outside the cell as beads. The key enzyme for the formation of PHA inclusions is the PHA synthase PhaC, which catalyses the polymerization of (R)- 3-hydroxyacyl-CoA to PHA with the concomitant release of CoA. The PHA synthase from Ralstonia eutropha (currently Cupriavidus necator), which is covalently bound to the PHA granule surface, tolerates fusions to its N terminus without loss of activity. In this study it was investigated if it would also tolerate translational fusions to its C terminus. A specially designed linker was employed, aiming at maintaining the hydrophobic surroundings of the R. eutropha synthase C terminus to allow proper folding and activity. Two reporter proteins were tested as fusion partners, the maltose binding protein MalE and the green fluorescent protein GFP. As GFP is a hydrophobic protein itself, no additional linker between the PHA synthase and the reporter protein was necessary to produce PHA granules displaying the functional fusion protein on the surface. Principally, the PHA synthase PhaC tolerates translational fusions to its C terminus but the nature of the fusion partner influences the functionality. Recently, PHA granules have often been acknowledged as bio-beads. A one-step production allows the formation of functionalised beads without the need for further cross-linking to impart desired surface properties. PHA beads displaying a gold- or silica-binding peptide at the N terminus of PhaC were constructed and tested for their applicability. Additionally, these beads were able to bind IgG due to the ZZ domain of the IgG binding protein A, which was employed as a linker sequence. These functionalised beads can be used as molecular tools in bioimaging and biomedicine, combining organic core with inorganic-binding shell structures. In a different biomimetic approach, the display of ten lysine residues at the granule surface was achieved using the phasin protein PhaP as the anchoring matrix. Extensive work was performed in an attempt to also employ the synthase protein, but was unsuccessful. These positively charged bio-beads can be used for dispersion or crosslinking experiments as well as silica binding.