Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Subject: search.filters.subject.Microbial exopolysaccharides

Search Results

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Investigation of the biosynthesis of exopolysaccharides within the biofilm matrix of Pseudomonas aeruginosa and Pseudomonas syringae pv. actinidiea : a thesis presented in partial fulfilment of the requirements for degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2017) Ghods, Shirin
    Polysaccharides are highly abundant natural biopolymers, which have biologically significant structural functions in living organisms. Various polysaccharides, with specific physicochemical properties, contribute to biofilm formation; defined as cell aggregations surrounded by extracellular polymeric substances. They are also important in the context of bacterial pathogenesis, while some have been harnessed for industrial and biomedical applications due to their unique chemical compositions and properties. In present study, we aimed at studying biofilm formation by Pseudomonas aeruginosa and P. syringae pv. actinidiae, respectively known as human and plant pathogens. In this context we focused on the production of exopolysaccharides, which predominantly constitute the biofilm matrix of these pathogenic bacteria. Here, we uncovered that the polysaccharide isolated from P. syringae pv. actinidiae biofilm mainly consists of rhamnose, fucose and glucose and it was cautiously introduced as a novel polysaccharide. In the context of disease control, and developing a management program, we provided some evidences for the effectiveness of chlorine dioxide and kasugamycin in the control of the bacteria living in both biofilm and planktonic modes. Furthermore, we investigated alginate biosynthesis as major polysaccharide contributing to mucoid biofilm formation by P. aeruginosa. We generated various mutants producing a variety of alginates with different chemical compositions. Also, this enabled us to analyse functional relationships of protein subunits involved in multiple steps of alginate biosynthesis including alginate polymerization, modification and secretion. We present evidence that while alginate unravelled that while alginate is polymerised and translocated across the membrane by a multiprotein complex, acetylation and epimerisation events positively and negatively correlated with the polymerization of the alginate or molecular mass, respectively. Analysis of the biofilms showed that biofilm architecture and cell-to-cell interactions were differently impacted by various compositions of the alginates. Also, this study provided insights into the c-di-GMP mediated activation of alginate polymerization upon binding to c-di-GMP as well as assigning functional roles to Alg8 and Alg44 including their subcellular localization and distribution. Here, we also used current knowledge of the alginate biosynthesis pathway to assess the production of alginate from biotechnologically accepted heterologous hosts including Escherichia coli and Bacillus megaterium strains. Primarily, we evaluated the production and functionality of the minimal protein requirements in nonpathogenic heterologous hosts, required for producing alginate precursor, and proceeding into polymerization and secretion steps. Overall, we concluded that polysaccharides play a major role in the formation of bacterial biofilms while chemical composition is a key determinant for biofilm architecture and development. This contribution to understanding the biosynthesis of bacterial polysaccharides and their properties could provide the necessary knowledge not only for developing novel therapeutics, but also for harnessing such biopolymers for various industrial applications and production via biotechnological procedures.
  • Thumbnail Image
    Item
    Incorporation of extracellular polysaccharide produced by Xanthomonas campestris into milk powders : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology at Massey University
    (Massey University, 2003) Sharpe, Hamish
    The purpose of the research was to investigate the functional properties of milk powders following exopolysaccharide (EPS) addition to milk solutions and their subsequent spray-drying. The aim was to replace some of the milk proteins with polysaccharide in dairy products while maintaining or improving the functional characteristics. Both commercial xanthan EPS and ferment xanthan EPS were incorporated into whole milk powder (WMP), skim milk powder (SMP), and milk protein concentrate (MPC). Ferment EPS was produced from a by-product of the dairy industry, milk permeate, through the hydrolysis of the lactose and fermentation with a strain of Xanthomonas campestris. Ferment EPS had a characteristic and unpleasant odour. The main compound responsible for this odour was p-cresol which, in milk, is largely bound in the conjugate form. Xanthomonas campestris hydrolyses these conjugates releasing the odour compounds. Ultrafiltration (UF) of the ferment or passing the ferment through a bed of activated carbon was effective in reducing the odour. UF was proven to reduce the levels of p-cresol in the ferment from 138ppb to less than 5ppb after 98 concentration factors. Milk powders made with UF ferment were more acceptable to the consumer sensory panel than those made with untreated ferment. The incorporation of EPS into milk powders has beneficial effects on the product with small additions increasing the viscosity of reconstituted SMP and WMP considerably. The EPS addition could result in a thickened milk product or alternatively, substitute for some of the milk solids. Sensory testing showed that 13.3% WMP solution, containing 0.02% commercial EPS, was not detectably different from a 15% WMP solution. The addition of both commercial and ferment EPS into milk powders leads to the formation of separate flocculated casein and polysaccharide phases with reconstituted milk. Confocal microscopy showed that casein flocculation occurred at all EPS concentrations tested, but this only resulted in sedimentation at intermediate EPS concentrations. At high EPS concentrations of approximately 0.2% the high viscosity limited flocculation and prevented sedimentation. At low EPS concentrations of approximately 0.05% flocculation was insufficient to overcome Brownian motion. Fresh cheese (Panela) made from MPC containing either ferment or commercial EPS showed greatly decreased whey loss. This was attributed to (i) the increased viscosity of the continuous phase limiting the flow of liquid through the pores of the cheese, and (ii) diminished casein interaction in the presence of EPS leading to a looser curd and lower contraction forces. For example the incorporation of 0.161% ferment EPS decreased the whey lost by approximately 75%. Negative effects were also apparent. The addition of EPS led to a granular appearance, which became more apparent with increasing EPS concentration. Cheese firmness was also decreased by approximately 40% by the addition of the ferment EPS at 0.161%. This could also be attributed to the localised aggregation of protein during renneting and the increased heterogeneity of the network. Sensory testing of cheeses made with 15.6% MPC + 0.045% commercial EPS compared with cheese made with 17.37% MPC alone showed that the consumers had no significant preference for one cheese over the other, but did notice a difference in texture. For reasons of safety and health, the sensory testing of milk and cheese in this research was confined to commercial xanthan. Future sensory testing of milk and cheese should be conducted with ferment EPS after odour removal rather than commercial EPS, and use consumers familiar with these cheese and milk products. For commercial production of dairy powders containing UF ferment EPS it is vital that either the xanthan or casein micelle structure be altered to prevent casein flocculation. If this is not feasible then an alternative use of the product may need to be found. A potential option involves the addition of the powder containing UF ferment EPS into food products as a minor food constituent. This may limit the occurrence of phase separation while improving the functionality of the product. Commercialisation is also limited by the increasing costs caused by ferment EPS purification and the lower solids concentrations required for spray-drying. As such the viability of the powder production must be determined.
  • Thumbnail Image
    Item
    Spatial and temporal localisation of exopolysaccharide gene expression in mucoid and non-mucoid Pseudomonas aeruginosa biofilms : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology, Massey University, Manawatu, New Zealand
    (Massey University, 2014) Holbrook, Stacey Ann Kashel
    The biofilm, or surface-associated microbial community, is the preferred method of growth for most bacteria. Pseudomonas aeruginosa is an ubiquitous, opportunistic pathogen capable of biofilm formation in a wide range of natural and clinical environments. In particular, biofilms formed by P. aeruginosa in the lungs of people with cystic fibrosis (CF) are responsible for a significant decline in the health and prognosis of these patients. Once established, P. aeruginosa biofilms begin to excrete an exopolysaccharide (EPS) called alginate which protects the bacterial microcolonies from antimicrobial molecules and confers a mucoid phenotype. Once this phenotypic switch has occurred, the biofilm becomes impossible to eradicate and ultimately leads to the death of the patient. Here, fluorescent signalling systems and confocal laser scanning microscopy (CLSM) have been used to spatially and temporally resolve the expression of three EPSs produced by P. aeruginosa; the pellicle-forming EPS (Pel), the EPS encoded by the polysaccharide synthesis locus (Psl) and alginate. In order to observe the effect (if any) of EPS production on spatial localisation of the cells within the biofilm, the biofilm-associated characteristics of three P. aeruginosa double-knockout mutants, each able to produce only one EPS has been observed. In analysing these biofilm structures, it was found that Pel has a role in facilitating an increased surface area of the biofilm, while Psl-producing mutants form a biofilm structure with a significantly increased biomass. By visualising fluorescent signals throughout a biofilm consisting of a mixture of the three mutants, the spatial localisation of EPS-producing bacterial populations has been observed. Here, Pel-producing mutants tended to aggregate at the attachment surface, suggesting a role in adhesion of the biofilm structure. Spatial and temporal localisation of EPS promoter activity was achieved by transforming the prototypic P. aeruginosa PAO1 strain with one of three plasmids encoding unstable gfp expression under the control of each EPS’s promoter sequence. Overall, this study has demonstrated the applications and limitations of fluorescence-based localisation of bacterial gene expression throughout P. aeruginosa biofilm development. Collectively, this information can help to guide future investigations into the expression and regulation of the genes associated with a biofilm phenotype, with the aim of identifying a target for effective therapy against this important pathogen.