“Green preservatives” – combating fungi in the food industry by applying antifungal lactic acid bacteria

Fungal spoilage is the most common type of microbial spoilage in food leading to significant economical and health problems throughout the world. Fermentation by lactic acid bacteria (LAB) is one of the oldest and most economical methods of producing and preserving food. Thus, LAB can be seen as an interesting tool in the development of novel bio-preservatives for food industry. The overall objective of this study was to demonstrate, that LAB can be used as a natural way to improve the shelf-life and safety of a wide range of food products. In the first part of the thesis, 116 LAB isolates were screened for their antifungal activity against four Aspergillus and Penicillium spp. commonly found in food. Approximately 83% of them showed antifungal activity, but only 1% showed a broad range antifungal activity against all tested fungi. The second approach was to apply LAB antifungal strains in production of food products with extended shelf-life. L. reuteri R29 strain was identified as having strong antifungal activity in vitro, as well as in sourdough bread against Aspergillus niger, Fusarium culmorum and Penicillium expansum. The ability of the strain to produce bread of good quality was also determined using standard baking tests. Another strain, L. amylovorus DSM19280, was also identified as having strong antifungal activity in vitro and in vivo. The strain was used as an adjunct culture in a Cheddar cheese model system and demonstrated the inhibition of P. expansum. Significantly, its presence had no detectable negative impact on cheese quality as determined by analysis of moisture, salt, pH, and primary and secondary proteolysis. L. brevis PS1 a further strain identified during the screening as very antifungal, showed activity in vitro against common Fusarium spp. and was used in the production of a novel functional wortbased alcohol-free beverage. Challenge tests performed with F. culmorum confirmed the effectiveness of the antifungal strain in vivo. The shelf-life of the beverage was extended significantly when compared to not inoculated wort sample. A range of antifungal compounds were identified for the 4 LAB strains, namely L. reuteri ee1p, L. reuteri R29, L. brevis PS1 and L. amylovorous DSM20531. The identification of the compounds was based on liquid chromatography interfaced to the mass spectrometer and PDA detector

Proteomic approaches to environmental stress in mussel Mytilus edulis due to emerging classes of anthropogenic pollutants

Anthropogenic pollutant chemicals pose a major threat to aquatic organisms. There is a need for more research on emerging categories of environmental chemicals such as nanomaterials, endocrine disruptors and pharmaceuticals. Proteomics offers options and advantages for early warning of alterations in environmental quality by detecting sub-lethal changes in sentinel species such as the mussel, Mytilus edulis. This thesis aimed to compare the potential of traditional biomarkers (such as enzyme activity measurement) and newer redox proteomic approaches. Environmental proteomics, especially a redox proteomics toolbox, may be a novel way to study pollutant effects on organisms which can also yield information on risks to human health. In particular, it can probe subtle biochemical changes at sub-lethal concentrations and thus offer novel insights to toxicity mechanisms. In the first instance, the present research involved a field-study in three stations in Cork Harbour, Ireland (Haulbowline, Ringaskiddy and Douglas) compared to an outharbour control site in Bantry Bay, Ireland. Then, further research was carried out to detect effects of anthropogenic pollution on selected chemicals. Diclofenac is an example of veterinary and human pharmaceuticals, an emerging category of chemical pollutants, with potential to cause serious toxicity to non-target organisms. A second chemical used for this study was copper which is a key source of contamination in marine ecosystems. Thirdly, bisphenol A is a major anthropogenic chemical mainly used in polycarbonate plastics manufacturing that is widespread in the environment. It is also suspected to be an endocrine disruptor. Effects on the gill, the principal feeding organ of mussels, were investigated in particular. Effects on digestive gland were also investigated to compare different outcomes from each tissue. Across the three anthropogenic chemicals studied (diclofenac, copper and bisphenol A), only diclofenac exposure did not show any significant difference towards glutathione transferase (GST) responses. Meanwhile, copper and bisphenol A significantly increased GST in gill. Glutathione reductase (GR) enzyme analysis revealed that all three chemicals have significant responses in gill. Catalase activity showed significant differences in digestive gland exposed to diclofenac and gills exposed to bisphenol A. This study focused then on application of redox proteomics; the study of the oxidative modification of proteins, to M. edulis. Thiol proteins were labelled with 5-iodoacetamidofluorescein prior to one-dimensional and two-dimensional electrophoresis. This clearly revealed some similarities on a portion of the redox proteome across chemical exposures indicating where toxicity mechanism may be common and where effects are unique to a single treatment. This thesis documents that proteomics is a robust tool to provide valuable insights into possible mechanisms of toxicity of anthropogenic contaminants in M. edulis. It is concluded that future research should focus on gill tissue, on protein thiols and on key individual proteins discovered in this study such as calreticulin and arginine kinase which have not previously been considered as biomarkers in aquatic toxicology prior to this study.