Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

Forest inventory-based large-scale forest biomass and carbon budget assessment: new enhanced methods and use of remote sensing for verification

In recent years, concern has arisen over the effects of increasing carbon dioxide (CO2) in the earth's atmosphere due to the burning of fossil fuels. One way to mitigate increase in atmospheric CO2 concentration and climate change is carbon sequestration to forest vegeta-tion through photosynthesis. Comparable regional scale estimates for the carbon balance of forests are therefore needed for scientific and political purposes. The aim of the present dissertation was to improve methods for quantifying and verifying inventory-based carbon pool estimates of the boreal forests in the mineral soils. Ongoing forest inventories provide a data based on statistically sounded sampling for estimating the level of carbon stocks and stock changes, but improved modelling tools and comparison of methods are still needed. In this dissertation, the entire inventory-based large-scale forest carbon stock assessment method was presented together with some separate methods for enhancing and comparing it. The enhancement methods presented here include ways to quantify the biomass of understorey vegetation as well as to estimate the litter production of needles and branches. In addition, the optical remote sensing method illustrated in this dis-sertation can be used to compare with independent data. The forest inventory-based large-scale carbon stock assessment method demonstrated here provided reliable carbon estimates when compared with independent data. Future ac-tivity to improve the accuracy of this method could consist of reducing the uncertainties regarding belowground biomass and litter production as well as the soil compartment. The methods developed will serve the needs for UNFCCC reporting and the reporting under the Kyoto Protocol. This method is principally intended for analysts or planners interested in quantifying carbon over extensive forest areas.

Gene flow from transgenic plant populations: models and applications for risk assessment

The future use of genetically modified (GM) plants in food, feed and biomass production requires a careful consideration of possible risks related to the unintended spread of trangenes into new habitats. This may occur via introgression of the transgene to conventional genotypes, due to cross-pollination, and via the invasion of GM plants to new habitats. Assessment of possible environmental impacts of GM plants requires estimation of the level of gene flow from a GM population. Furthermore, management measures for reducing gene flow from GM populations are needed in order to prevent possible unwanted effects of transgenes on ecosystems. This work develops modeling tools for estimating gene flow from GM plant populations in boreal environments and for investigating the mechanisms of the gene flow process. To describe spatial dimensions of the gene flow, dispersal models are developed for the local and regional scale spread of pollen grains and seeds, with special emphasis on wind dispersal. This study provides tools for describing cross-pollination between GM and conventional populations and for estimating the levels of transgenic contamination of the conventional crops. For perennial populations, a modeling framework describing the dynamics of plants and genotypes is developed, in order to estimate the gene flow process over a sequence of years. The dispersal of airborne pollen and seeds cannot be easily controlled, and small amounts of these particles are likely to disperse over long distances. Wind dispersal processes are highly stochastic due to variation in atmospheric conditions, so that there may be considerable variation between individual dispersal patterns. This, in turn, is reflected to the large amount of variation in annual levels of cross-pollination between GM and conventional populations. Even though land-use practices have effects on the average levels of cross-pollination between GM and conventional fields, the level of transgenic contamination of a conventional crop remains highly stochastic. The demographic effects of a transgene have impacts on the establishment of trangenic plants amongst conventional genotypes of the same species. If the transgene gives a plant a considerable fitness advantage in comparison to conventional genotypes, the spread of transgenes to conventional population can be strongly increased. In such cases, dominance of the transgene considerably increases gene flow from GM to conventional populations, due to the enhanced fitness of heterozygous hybrids. The fitness of GM plants in conventional populations can be reduced by linking the selectively favoured primary transgene to a disfavoured mitigation transgene. Recombination between these transgenes is a major risk related to this technique, especially because it tends to take place amongst the conventional genotypes and thus promotes the establishment of invasive transgenic plants in conventional populations.

Agricultural expansion and climate change in the Taita Hills, Kenya: an assessment of potential environmental impacts

The indigenous cloud forests in the Taita Hills have suffered substantial degradation for several centuries due to agricultural expansion. Currently, only 1% of the original forested area remains preserved in this region. Furthermore, climate change imposes an imminent threat for local economy and environmental sustainability. In such circumstances, elaborating tools to conciliate socioeconomic growth and natural resources conservation is an enormous challenge. This dissertation tackles essential aspects for understanding the ongoing agricultural activities in the Taita Hills and their potential environmental consequences in the future. Initially, alternative methods were designed to improve our understanding of the ongoing agricultural activities. Namely, methods for agricultural survey planning and to estimate evapotranspiration were evaluated, taking into account a number of limitations regarding data and resources availability. Next, this dissertation evaluates how upcoming agricultural expansion, together with climate change, will affect the natural resources in the Taita Hills up to the year 2030. The driving forces of agricultural expansion in the region were identified as aiming to delineate future landscape scenarios and evaluate potential impacts from the soil and water conservation point of view. In order to investigate these issues and answer the research questions, this dissertation combined state of the art modelling tools with renowned statistical methods. The results indicate that, if current trends persist, agricultural areas will occupy roughly 60% of the study area by 2030. Although the simulated land use changes will certainly increase soil erosion figures, new croplands are likely to come up predominantly in the lowlands, which comprise areas with lower soil erosion potential. By 2030, rainfall erosivity is likely to increase during April and November due to climate change. Finally, this thesis addressed the potential impacts of agricultural expansion and climate changes on Irrigation Water Requirements (IWR), which is considered another major issue in the context of the relations between land use and climate. Although the simulations indicate that climate change will likely increase annual volumes of rainfall during the following decades, IWR will continue to increase due to agricultural expansion. By 2030, new cropland areas may cause an increase of approximately 40% in the annual volume of water necessary for irrigation.

Assessment of In-House Natural Product and Synthetic Compound Libraries Based on In vitro Inhibition of Cholinesterases

The first line medication for mild to moderate Alzheimer s disease (AD) is based on cholinesterase inhibitors which prolong the effect of the neurotransmitter acetylcholine in cholinergic nerve synapses which relieves the symptoms of the disease. Implications of cholinesterases involvement in disease modifying processes has increased interest in this research area. The drug discovery and development process is a long and expensive process that takes on average 13.5 years and costs approximately 0.9 billion US dollars. Drug attritions in the clinical phases are common due to several reasons, e.g., poor bioavailability of compounds leading to low efficacy or toxic effects. Thus, improvements in the early drug discovery process are needed to create highly potent non-toxic compounds with predicted drug-like properties. Nature has been a good source for the discovery of new medicines accounting for around half of the new drugs approved to market during the last three decades. These compounds are direct isolates from the nature, their synthetic derivatives or natural mimics. Synthetic chemistry is an alternative way to produce compounds for drug discovery purposes. Both sources have pros and cons. The screening of new bioactive compounds in vitro is based on assaying compound libraries against targets. Assay set-up has to be adapted and validated for each screen to produce high quality data. Depending on the size of the library, miniaturization and automation are often requirements to reduce solvent and compound amounts and fasten the process. In this contribution, natural extract, natural pure compound and synthetic compound libraries were assessed as sources for new bioactive compounds. The libraries were screened primarily for acetylcholinesterase inhibitory effect and secondarily for butyrylcholinesterase inhibitory effect. To be able to screen the libraries, two assays were evaluated as screening tools and adapted to be compatible with special features of each library. The assays were validated to create high quality data. Cholinesterase inhibitors with various potencies and selectivity were found in natural product and synthetic compound libraries which indicates that the two sources complement each other. It is acknowledged that natural compounds differ structurally from compounds in synthetic compound libraries which further support the view of complementation especially if a high diversity of structures is the criterion for selection of compounds in a library.