The consumption of meat is increasingly linked to various diseases and this has already affected the growth of this sector of the food industry in some countries. Pork is seen as one of the major contributors to this problem. The meat industry reacted by using strategies such as dietary supplementation and direct addition of healthier lipids to manipulate the nutritional value of meat. The positive effects of CLA on human health are well documented and various strategies have been successfully employed in increasing the levels of CLA in different animal models such as pigs and eventually pork products. The effects CLA may have on a fermented meat product like salami has not been studied yet. No research have been reported where it was attempted to increase the nutritional value of salami, maintain acceptable product quality and include a therapeutically high level of CLA with the belief that it will benefit human health. In the first experiment of this study, 40 Duroc X Landrace gilts weighing on average 35 kg were randomly divided into two groups fed either a diet containing 0.5% sunflower oil (SFO) or a diet containing 0.5% conjugated linoleic acid (Luta-CLA® 60, BASF). These groups were further divided into two slaughter weight groups of ±70 kg and ±90 kg. After slaughter the lean meat and backfat from the loins of these animals were pooled by treatment group and utilized to manufacture salami. The aim was to determine if salami quality is influenced by slaughter weight and dietary supplementation of CLA. Both variables had major effects on the fatty acid composition and fatty acid ratios of the muscle and fat raw material as well as salami. The fatty acids and fatty acid ratios of technological importance were mostly positively influenced while the fatty acids and fatty acid ratios of nutritional and health concern were mostly negatively influenced by increased slaughter weight and dietary CLA supplementation. The microbial, physical, sensory and lipid stability parameters of salami were unaffected or inconsistently affected by both variables. Although dietary CLA was deposited successfully in muscle and fat, the deposition level was low. Consumption of a 28 g portion of salami manufactured from CLA enriched pork could only supply in 1% of the RDA for CLA. It could be concluded that although dietary supplementation of pork with CLA improved the technological properties of fat tissue it could not be considered a very successful approach to increase human consumption of CLA. In the second experiment of this study the aim was to increase the CLA content of salami to three different percentages (25%, 50% and 100%) of the RDA for CLA per 28 g portion of salami. This was accomplished through the direct addition of CLA (Tonalin® TG 80) in a pre-emulsified form with proportional decreases in the normally used pork BF content of the salamis. The salamis from these three treatment groups were then compared to a 100% pork BF control group for any possible effects on the microbial, physical and lipid stability parameters as well as fatty acid composition and fatty acid ratios. Microbial and sensory parameters were largely unaffected with varying effects on the physical and lipid stability parameters. Major effects on the fatty acid composition and fatty acid ratios of the salamis were observed. The partial replacement of pork BF and direct addition of CLA to salami proved to be an effective method of increasing CLA levels in salami in an attempt to improve the health aspects of salami to the point where it could be regarded as a functional food.

Wetlands account for approximately 20% of annual global methane emissions. Many wetlands receive inputs of organic matter, nutrients, metals and various toxic compounds from adjacent agricultural and industrial areas. The present study aimed to investigate the microbial composition of a natural methanogenic consortium. A consortium-based molecular approach to study diversity of methanogenic microbial communities in a natural wetland at the primary inflow was used. Key microorganisms of a nethane producing consortium were identified. Extracted high molecular mss DNA ws analysed by PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rDNA. This study was also aimed to identify syntrophic microorganisms in the wetland system. The data obtained suggest a well established syntrophic relationship within the wetland.

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This study characterised the bacterial flora of a commercially produced Stilton cheese in an effort to determine the contribution of non-starter lactic acid bacteria (NSLAB) to its aroma profile. A total of 123 microbial strains previously isolated from different sites (outer crust, blue veins and white core) of the cheese sample obtained at the end of ripening (~8 weeks) were recovered in MRS and BHI broths and preliminarily identified using conventional microbiological methods in order to establish population diversity and to screen out yeasts and moulds. Organisms identified with partial 16S rDNA sequence analysis were Lactobacillus plantarum, Lactobacillus brevis, Enterococcus faecalis, Staphylococcus aureus, Acinetobacter baumanii and Psychrobacter spp., with the genus Lactobacillus being the dominant (75%) group detected in all the sampled sites. Cluster analysis of pulse-field gel electrophoresis patterns associated the Lactobacillus isolates according to their site of isolation. Lb. plantarum isolates, two from each of the cheese sites, were evaluated for tolerance to heat stress and to different levels of salt, acid and relative humidity (RH) in order to ascertain whether the stress conditions associated with the isolation site could select the phenotype of microbial species recovered. The D72°C values revealed that isolates obtained from the outer crust were more heat sensitive suggesting they may have colonised the cheese post-pasteurisation. All the isolates were sensitive at pH range 3-4 but could grow at pH range 4.5-5. Similarly, isolates could grow at 3.5-5% (w/v) sodium chloride but were suppressed at 10%. Lactobacilli from the outer crust were the most halo-tolerant growing at 8% sodium chloride. For all strains, survival was low at 33-54% RH when cells were suspended in sterile de-ionised water but survived better at 33% RH in maximum recovery diluent (MRD) suggesting cellular protection by MRD. Lb. plantarum isolates from each site (outer crust=7; blue veins=19; white core=24) were tested for antimicrobial activity against Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Staph. aureus, Salmonella Typhimurium, Clostridium sporogenes, Lb. pentosus and Lactococcus lactis using the plate agar overlay and paper disc diffusion assays. All the 59 Lactobacillus isolates were tested for plantaricin EF genes using PCR. The nature of antimicrobial activity was examined using cell-free supernatants treated to neutralise acids and/or hydrogen peroxide. Treatment with proteinase K was used to ascertain whether activity was due to bacteriocin (putative plantaricin) production. On solid medium, the isolates had antimicrobial activity against Gram-negative and Gram-positive bacteria, each isolate showing activity against more than one species. Lb. pentosus, Ps. aeruginosa, E. coli and L. monocytogenes were the most sensitive whereas Cl. sporogenes was the most resistant spp. Activity against these organisms was mainly attributed to acid, and to a less extent, hydrogen peroxide and plantaricin production. Whereas Lb. plantarum isolates had a high prevalence of plantaricin EF genes, there was weak evidence for plantaricin production in liquid medium assays. Plantaricin production was only demonstrable among Lb. plantarum isolates from the veins and core against Lb. pentosus, implying the phenomenon was largely dependent on the genotype/strain of Lb. plantarum and was only active against closely related lactic acid bacteria. Subsequently, the effect of growth and survival dynamics of the different genotypes of the organism on the volatile aroma profiles of milk was examined. Individual isolates, one from each of the cheese sites, were co-cultured with acid-producing Lc. lactis (APL) and non acid-producing Lc. lactis (NAPL) in UHT milk under simulated cheese ripening conditions. During early fermentation (0-48 h, 30oC), the isolate obtained from the blue veins stimulated more growth of Lactococcus strains in mixed culture when compared to single cultures and to Lactobacillus isolates obtained from other sites in mixed culture. The volatile profiles of all Lb. plantarum strains grown alone were not significantly different (p>0.05). The type and levels of volatiles detected in mixed culture depended on the genotype/strain of Lb. plantarum inoculated as well as the acidification capability of Lc. lactis with which it was co-cultured. Co-culture of Lactobacillus isolates with APL resulted in increased aldehyde and alcohol production, whereas with NAPL only acetoin synthesis was enhanced. Salt addition had minimal effect on the volatile profiles. During further incubation (12 weeks, 18oC), growth of Lb. plantarum strains was better in salted samples inoculated with NAPL. The NAPL strain remained stable at 7 log10 CFU/ml throughout, while the APL rapidly declined from 9 to less than 5 log10 CFU/ml. The highest level of alcohols, organic acids and acetoin was detected from samples inoculated with the pure culture of the Lactobacillus isolate obtained from the blue veins. Co-culture of the isolate with APL enhanced acid and alcohol production, whereas its co-inoculation with NAPL increased acetoin synthesis. As Lb. plantarum is an incidental organism in cheese, its presence is unpredictable; it was therefore concluded that occurrence of different genotypes of the organism could be a major contributory factor to the variations in the cheese quality characteristics from batch to batch.

In the blue cheese Stilton the starter mould Penicillium roqueforti grows and sporulates during the ripening period and is considered to be responsible for the unique blue cheese aroma. However, the sporulation of the mould, which results in the formation of blue veins, takes place in a fraction of the Stilton matrix which overall is very heterogeneous. Most blue cheeses develop a secondary microflora of yeasts which may affect their aroma. The aim of this study was to investigate the yeast flora of Stilton, the aroma profile of the cheese and the role of the yeasts in the aroma production. The approach in this work was to study individually the different sections of Stilton (the blue veins, the white core and the outer crust) as previous studies have demonstrated each section has a differing bacterial flora. In addition to the classical microbiology, a series of molecular techniques (Denaturing Gradient Gel Electrophoresis, Restriction Fragment Length Polymorphism and Terminal RFLP) were compared and applied for the screening of the local fungal communities in the cheese. The results showed that the two approaches were complementary. It was concluded that the structure of the fungal community was different for each section of the cheese. The aroma profiles of the three different sections of Stilton were studied using solvent extraction Gas Chromatography-Mass Spectrometry (GC-MS), a headspace GC-MS technique (SPME GC-MS) and direct headspace analysis (Atmospheric Pressure Chemical Ionisation [APCI]-MS). The different sections of Stilton presented different aroma profiles. Overall, the blue and the outer crust had similar profiles. These two sections contained higher amount of ketones while the white contained higher amounts of alcohols and aldehydes. Yeast isolates and the starter Penicillium roqueforti were cultivated alone and in combination in a cheese model and the aroma production was studied with SPME GC-MS analysis. The co-culture of the starter Penicillium roqueforti and individual yeast isolates resulted in aroma profiles different from those that were produced by the mould or the yeasts individually. The model of Penicillium roqueforti with Yarrowia lipolytica resulted in an aroma more similar to blue cheese than produced by the mould alone. Sensory analysis (Flash profile technique) was used in order to compare the aroma of this model with the aroma of blue cheeses and the perception of the combined culture was found to be similar to Stilton cheese, whereas that of the mould alone was not. Yeasts are a significant part of the microflora of Stilton and they are able to affect the aroma production. Selected isolates of Yarrowia lipolytica could be used in combination with Penicillium roqueforti for the production of blue cheese aroma e.g. as a starter culture.

CCL3LI and CCL4LI are chemokine genes, located on chromosome 17q12. They are copy number variable genes which share 95% sequence identity with their non-copy number variable paralogues CCL3 and CCL4. The copy number of these genes varies between populations and has been reported to be associated with phenotypes such as susceptibility to HIV infection, hepatitis C virus infection, Kawasaki disease and SLE. The aim of this study is to understand the evolutionary history of variation at the CCL3L1/CCL4LI cluster. To accomplish this goal, several approaches including typing microsatellites, single nucleotide polymorphisms (SNPs) and CCL3L 1/CCL4L 1 sequence haplotypes were used to investigate the association with CCL3L 1 and CCL4L 1 copy number. However, the results showed that there is no strong association between a single-copy marker and CCL3L 1 and CCL4LI copy number, but there is evidence of recombination. Therefore, this may suggest that CCL3L 1/CCL4L 1 is a complex region and one plausible hypothesis is that there is a high rate of recombination in this region. This study of the evolution of CCL3L 1/CCL4L 1 haplotypes showed that a major one-copy CCL3L 1/CCL4L I haplotype (about 70% haplotype frequency) identified in humans, represents the ancestral state, as inferred from comparison with chimpanzee.

<p>Although there are many examples of microbial biogeography, few microbes have been studied at high taxonomic resolution over large spatial scales. As a result, the environmental and ecological processes that drive niche partitioning, diversity, composition, and activity of microbial taxa are often poorly understood. To address this gap, I examine the most abundant phytoplankton in the global ocean, Prochlorococcus sp., a marine cyanobacterium. Using amplicon libraries of the Prochlorococcus internal transcribed spacer (ITS) region and 23S rRNA gene as markers, I demonstrate several key differences between the two major high light (HL) clades of Prochlorococcus. First, by examining ITS amplicon libraries at high taxonomic resolution it is revealed that “sub-ecotype” clades have unique, cohesive responses to environmental variables and distinct biogeographies, suggesting that presently defined ecotypes can be further partitioned into ecologically meaningful units. Whereas unique combinations of environmental traits drive the distribution of the HL-I sub-ecotype clades, the HL-II sub-ecotype clades appear ecologically coherent. Second, using 23S rRNA and rDNA libraries I show that activity (rRNA) and abundance (rDNA) are highly correlated for Prochlorococcus across all sites and operational taxonomic units (OTUs) in the surface ocean, demonstrating a tight coupling between activity and abundance. Finally, I investigate the associations between Prochlorococcus and the rest of the microbial community in the North Pacific and find region-specific trends in both strength and sign. Associations with other microbes are strongest for HL-I in the temperate region and strongest for HL-II in the sub-tropical gyre. This dissertation clarifies the relative importance of the environment, geography, community, and taxonomy in terms of their role in creating complex assemblages of Prochlorococcus and helps improve our understanding of how marine microbial communities are assembled in situ.</p> / Dissertation