Lactation provides the singular source of nourishment to the offspring of mammals. This nutrition source also contains a diverse microbiota affecting the development and health of the newborn. Here, we examined the milk microbiota in water deer (Hydropotes inermis, the most primitive member of the family Cervidae), reindeer (Rangifer tarandus, the oldest semi-domesticated cervid), and the dairy goat (Capra aegagrus, member of the family Bovidae), to determine if common milk microbiota species were present across all three ruminant species. The results showed that water deer had the highest bacterial diversity, followed by reindeer, and then goat. Unifrac distance and correspondence analyses revealed that water deer harbored an increased abundance of Pseudomonas spp. and Acinetobacter spp., while milk from reindeer and goat was dominated by unclassified bacteria from the family Hyphomicrobiaceae and Bacillus spp., respectively. These data indicate significant differences in the composition of milk-based bacterial communities. The presence of Halomonas spp. in three distinct co-occurrence networks of bacterial interactions revealed both common and unique features in milk niches. These results suggest that the milk of water deer and reindeer harbor unique bacterial communities compared with the goat, which might reflect host microbial adaptation caused by evolution.

Plants are a major force shaping how ecosystems function, including non-native plants. The effects that non-native plants have on ecosystem properties and processes can be particularly important as invasive plants are associated with changes in nitrogen and carbon cycling Plants can also indirectly affect ecosystem processes through their influence on the soil microbial community and different plants are associated with different microbial communities. The legacies produced by invasive plants can be long-lasting and inhibit the restoration of damaged ecosystems. Because of the central role of soil fungi in ecosystem processes, I examined how fungal abundance and community composition were altered by non-native plants, and the persistence of these changes. Specifically, I examined how two different cases of invasion by non-native species affected soil fungi over three years compared to soil fungi in native, undisturbed sites. I further tested how the soil fungi responded to the removal of the non-native plants and to inoculation with local native microbial communities. Legacy effects of land use history on soil fungal abundance and community composition were found in these central Florida communities. There were substantial differences in soil fungal abundance and community composition in disturbed and pasture sites compared to native scrub, and these differences persisted for three years after non-native grasses were removed. Not only did the grass-dominated pasture and disturbed sites differ from the undisturbed native shrub-dominated ecosystem, they differed significantly from each other, indicating that the different non-native grasses and other specific changes associated with each land use played a role in soil fungal communities. The combined results of this study have implications for restoration ecology. The current dependence of the fungal community on land use and the associated non-native species invasions (along with other analyses done in this system) suggest that a different approach to restoration is required here to overcome the observed legacy effects. / text

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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.

The overall aim of this study was to eliminate the root cause of the ‘cheese flavour’ in spread caused by four key microbes Bacillus spp, Staphylococcus spp, yeasts and moulds. The major sources of these bacteria were in the product ingredients mainly sweet cream buttermilk and skimmed milk along with environmental aerosols. The causative organisms were present in about 63% of the products and mainly ‘feed’ on the oil element of the recipe, containing high level C12 that generates the distinctive cheese flavour when broken down by bacteria. The key hurdle factor in spread preventing microbial growth is water droplet size. The spread showing cheese off flavour had a droplet size distribution of 95% <10 micron. To achieve finer droplet size distribution, trial products were made in the Scrape Surface Heat Exchanger (SSHE) over the current churning method with a distribution of droplet size 98% <5 micron. The trial product showed a 50% reduction in the generation of the ‘cheese flavour’ methyl ketones. The Staphylococcus spp cross contamination source where from personnel with direct food contact processing area. Further education on personal hygiene helped to reduce the level of Staphylococcus spp contamination in the product. The trial product from the SSHE was further challenge tested with Listeria monocytogenes over a 10 week shelf life period to evaluate product robustness against microbial growth and spoilage. The organism did not show any growth over the period of time. The liquid phase of the emulsion was further modified with various salts at different concentrations and challenged with L. monocytogenes isolated from various parts of the dairy environment. It was observed that a pH range of 5.5 or lower with added 0.063% potassium sorbate showed significant antibacterial affect compared to the nutrient enriched MPC-broth and the unsalted liquid phase of the emulsion with no added potassium sorbate. To understand L. monocytogenes survival within a dairy process, the organism was further challenged by exposure to pasteurisation heat treatments and the standard CIP cycle of acid and caustic treatment. No recovery rate of the organism was observed. Therefore it could be concluded that the contamination within the industry is more likely to be post process or environmental contamination rather than survival through the plant itself as per RASFF alert of Listeria spp outbreak in dairy. Therefore, reducing the available water in the liquid phase of the spread and achieving a <5 μm droplet size and a finer distribution within the product will be limiting factors to microbial growth. An air purifier system BAXX has reduced the level of environmental contaminants, especially yeast and mould.