The age of Aspergillus parasiticus (1-11-105Wh1) mycelium was found to have an influence on the level of enzymes, responsible for the conversion of sterigmatocystin to aflatoxin B[1] and O-methylsterigmatocystin, present. These enzymes were active over a wide range of temperature and pH. Production of a cell free system by lyophiliization yielded the highest aflatoxin B[1] synthesising activity. Three other methods of preparing the cell free system capable of synthesising aflatoxin B[1] were also studied, ie,: french press, protoplast, and grinding, but with limited success. The lyophilized preparation had narrower temperature and pH optima for the conversion than whole mycelia. Initial purification of the aflatoxin B[1] synthesising enzyme was achieved by separating the crude cell free extract by gel filtration. The enzyme activity was located in a membrane fraction. The involvement of endoplasmic reticulum was indirectly concluded by the use of marker enzyme and chelating agents. This membrane fraction was ultracentrifuged and the released extrinsic proteins were separated by gel filtration. A fraction containing two proteins which were capable of converting sterigmatocystin to aflatoxin B[1] was isolated and characterised by isoelectric focusing and gel electrophoresis. The temperature and pH optima together with the cofactor requirements were studied. The Michaelis-Menten constant (Km) and the stoichiometry for the conversion of sterigmatocystin to aflatoxin B[1] was determined. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1984.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed Feb. 20, 2008). PDF text: VI, iii, 335 p. : ill. ; 15 Mb. UMI publication number: AAT 3274813. Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 112-120). Also issued on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / "July 2004." Typescript. Vita. Includes bibliographical references (leaves 112-120). Also issued on the Internet.

Thesis (M. Sc.)--Carleton University, 1989. / Also available in electronic format on the Internet.

M.Tech. / A variety of fungal species belonging to the genera Aspergillus, Penicillium, Fusarium and Alternaria, Cladosporium are common contaminants of food commodities such as grains, cereals, seeds, nuts, and fruits. These fungal species in turn produce mycotoxins under favourable conditions as secondary metabolites, which have been recorded to have harmful effects in both animals and man. The concern for mycotoxin contamination of food commodities grown and produced in sub-Saharan Africa has grown considerably over the years with increased and improved strategies on mycotoxin monitoring. The aim of this project was to determine and evaluate the quality of food commodities grown and produced in selected rural areas of the southern part of Nigeria in relation to fungi and mycotoxins, evaluate the health implications of the mycotoxins, as well as, suggest possible solutions to reduce exposure of the population in these areas to fungi and mycotoxins in the food. This study was of much importance as the populace of these areas depend solely on the food commodities grown and produced in these areas. Ochratoxin A, aflatoxins, deoxynivalenol, fumonisins and zearalenone are major mycotoxins occurring naturally in most food commodities that have important health significances and as such, it was important to determine the exposure of people in this part of the country to these major mycotoxins. A total of 144 samples comprising of rice (41), maize (39), cocoa (39) and cocoa-based powder beverage (25) collected from the fields, markets and stores were screened for Aspergillus, Fusarium and Penicillium species producing mycotoxins using conventional methods and DNA sequencing which were preceded by serial dilution of samples on agar plates. Further analysis for mycotoxin extraction was done using multi-mycotoxin extraction, strong anion exchange columns and immunoaffinity columns, which was followed by identification and quantification via thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Mycological screening of samples showed incidences of various species of filamentous fungi including A. flavus, A. parasiticus, A. ochraceus, A. niger, F. verticillioides, F. proliferatum and F. graminearum, with highest incidences of A. flavus in rice (65.9%), F. verticillioides in maize (76.9%), A. flavus in cocoa (77.8%) and Cladosporium in cocoa-based powder beverages (32%). Mycotoxicological analysis showed occurrences of aflatoxins, ochratoxin A, zearalenone, fumonisins and deoxynivalenol in all food samples analysed with fumonisins, aflatoxins, ochratoxin A and deoxynivalenol prevalent in maize samples; ochratoxin A, aflatoxins and fumonisisn prevalent in rice samples; aflatoxins, ochratoxin A and fumonisins prevalent in cocoa samples and ochratoxin A, fumonisins and zearalenone prevalent in cocoa-based powder beverage samples. Levels of mycotoxin contamination varied from field to market place to storage with higher contamination of mycotoxins in samples from markets and store houses. In order to determine the health implications of the extracted mycotoxins, cytotoxicity analysis was done using MTT (methylthiozol tetrazolium)- assay on human blood lymphocytes and results showed a reduction in cell viabilities on cells exposed to extracts contaminated with mycotoxins at varying concentrations over 24hrs and 48hrs duration. It was seen that although levels of mycotoxins in samples were below and some above the set regulatory limits of mycotoxins in food, daily exposure to these mycotoxins over a long period of time could be dangerous health-wise influencing symptoms such as immunosuppression, kidney disorders, reproductive health disorders, liver and oesophageal cancers. It is therefore necessary to educate rural population and other populace on the health implications of ingesting these mycotoxins as well as teach them simple and cheap methods of preventing mycotoxin contamination of food commodities.

M.Tech. / Maize (Zea may) is the third most important cereal in the world serving various purpose of economic importance especially as staple diet to the Africans and as a major component of animal feed. Unfortunately, this commodity serves as a suitable substrate for pest and fungi development which may result in the production of mycotoxins. Mycotoxins are secondary metabolites of varying chemical structures produced by filamentous fungi, which may contaminate agricultural commodities either in the field or at storage. Mycotoxins have been implicated to cause several diseases in humans and animals ranging from acute to chronic. This study was designed to determine and quantify the occurrence and levels of mycotoxins in South African feed grade commercial maize. A total of 40 commercial feed grade maize samples were randomly sampled from two factory sites (Factory A and Factory B) of a commercial feed company source from known South African maize producers. The samples were screened for fungi using the conventional method while the mycotoxin screening and quantification was done by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). In addition to TLC and HPLC, VICAM/HPLC, VICAM/fluorometer, enzyme linked immuno-sorbent assay (ELISA) and lateral flow method were used for determination and quantification of fumonisins. The mycological investigation revealed the occurrence of several fungal species of Fusarium, Aspergillus and Penicillium with Fusarium being the most prevalent (100%). Among the Fusarium spp. were F. verticillioides, F. proliferatum, F. oxysporum and F. graminearum, F. subglutinans, F. chlamydosporum, F. solani, F. poae and F. dimerum. Fusarium verticillioides and F. proliferatum had the highest incidence rate of 89% and 73%, respectively, followed by F. oxysporum (65%) and F. graminearum (48%). The rate of occurrence of A. fumigatus, A. flavus and Penicillium spp. were 45%, 43% and 38%, respectively. Further analysis on the isolated fungal strains proved that over 50% of the fungal spp. were toxigenic. Mycotoxicological study on the samples revealed that the samples were contaminated with fumonisins (FB), aflatoxins B (AFB), ochratoxin A (OTA) and zearalenone (ZEA) on TLC. Mycotoxins levels in the samples were confirmed on HPLC, with the levels ranging from 0.064-1.035 ppm for FB, 0-0.762 ppm for iii AFB, 0-0.194 ppm for OTA and 0-0.135 ppm for ZEA. There was no significance difference on mycotoxin concentration in the feed grade sample between the two factory sites. Results obtained from the VICAM methods, ELISA and lateral flow method for FB determination showed 100% incidence rate of the feed grade maize samples similar to that observed on HPLC. Although ELISA (0.190-2.450 ppm) and lateral flow method (0.350-2.700 ppm) showed higher concentration of FB compared to HPLC (0.064-1.035 ppm), recovery analysis on sample using lateral flow gave high value (85%) comparable to HPLC. Further analyses were performed to determine the cytotoxic potential of the feed grade maize samples using the fumonisin fraction (FFraction). The data obtained showed that the feed grade maize extracts were toxic on human peripheral blood mononuclear cells (HPBMC) and the degree of effect on the cells is dependent on the concentration of FB and the duration of exposure. However using oneway ANOVA to determine the effect of different concentration (volumes) of toxin and time of exposure on HPBMC in this study, it was observed that there was no significant difference regardless of the concentration and time.

Guided by increasing legislation, the analysis of food borne toxins, including mycotoxins, seeks to address market related demands for the development of analytical systems to monitor this threat to food security and human health. This Thesis is directed at the assessment of the application of electrochemistry for direct electroanalysis and characterisation of the mycotoxin citrinin (CIT) in aqueous media as well as fundamental investigations of the surface of polished and oxidised glassy carbon electrodes (GCE). This study provides the first known account of CIT detection through electrochemical methods. Although electrochemically active, CIT current responses (Ip) were highly irreproducible at polished GCE with a coefficient of variation (C.V.) of 20.16 %. As stability of Ip across multiple electrode preparations is a key requirement in electroanalysis, investigations were directed at attaining stability in CIT Ip. Achieving stability in CIT Ip was investigated via two approaches, including: accounting for Ip variability between electrode preparations as a result of variable GCE surface conditions as a post-data-acquisition analysis and secondly, removing Ip variability through modification of GCE. Accounting for variability in Ip was investigated through the application of double layer capacitance as an indicator of the activity of an electrode, and in so doing serving as a relative mediator of Ip responses between electrodes. Application of this procedure dropped CIT C.V. to a third of starting value across polished GCE (C.V. = 7.18 %), chemically oxidised GCE (Pi-GCE, C.V = 8.47 %) and functionalised multi-walled carbon nanotube modified GCE (fMWCNT, C.V. = 25.79 %) and was effective with analysis of structurally distinct molecules, 2,4-dimethylaniline (2,4-DMA) and 1,2,4-trihydroxybenzene (Triol). Furthermore, it afforded the ability to determine discreet solution overlapping data sets of Ip. Stabilising Ip through GCE surface modification was achieved by anodic electro-oxidation of GCE and allowed for direct electroanalysis of CIT and subsequent characterisation and analysis of CIT in complex media as it reduced C.V. of CIT Ip to 0.73 %. Fundamental investigations of the electrode surface condition are described such that the source of variability could be identified and the interactions of CIT with the electrode understood. Two surface oxidation techniques were applied in modification of GCE; anodic electro-oxidation (EOx GCE) and chemical oxidation using piranha solution (Pi-GCE), analysis of which has previously not been reported. Fundamental analyses to determine surface morphology and chemistry of Pi-GCE, EOx-GCE and polished GCE were conducted using high resolution scanning electron microscopy (HRSEM), scanning electrochemical microscopy (SECM), energy dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), fourier transform infrared spectroscopy (FTIR) and via electroanalytical methods. These studies showed that both oxidation procedures introduced a variety of oxide species at GCE surface, and further that the extent of those species was similar with total % O being 27.67 % and 33.47 % at Pi-GCE and EOx-GCE respectively. Although chemically similar, each surface was morphologically distinct. Electrochemical analyses at the surfaces revealed Pi-GCE to behave more similarly to polished GCE than EOx-GCE. As CIT responses were found to be stable at EOx-GCE (C.V. = 0.73 %) as opposed to Pi-GCE (C.V. = 22.87 %), stability of CIT Ip was likely to be as a result of a physical interaction with electrode morphology rather than interaction on a chemical basis. Morphological analyses revealed polished GCE and Pi-GCE to be highly morphologically irregular at the micro-scale. Although comparatively smooth, the surface morphology of EOx-GCE does not account for the stability of Ip. This study thus proposed a theory to describe the mechanism by which the limited conductivity and porosity of EOx-GCE allow for it to provide a relatively stable surface area within the oxide layer, adjacent to the electrode surface, and thus provided a stable platform for electroanalysis. Voltammetric characterization of CIT at EOx-GCE revealed that anodic oxidation in aqueous media involved an uneven number of electrons to protons via an ECE mechanism. This was illustrated to be nt = 2e- accompanied by the transfer of 1H⁺ per molecule oxidised. A proposed reaction scheme for the initial stages of CIT oxidation was suggested to involve both hydroxyl and carboxyl moieties of the CIT molecule. CIT oxidation was shown to arise as a result of a relatively complex mass transport regime which included both adsorptive and diffusive derived Ip₁. The LOD in buffered aqueous media was found to be 16 nM, a highly competitive result in relation to chromatographic techniques. Further application of EOx-GCE in complex media illustrated that CIT associates non-specifically with the components of food samples, primarily proteins. As a result of this, extraction of CIT from such media is mandatory. Liquid-liquid extraction illustrated a recovery in CIT Ip₁ and in so doing provided a means of accurately and sensitively detecting CIT from food samples with an LOD of 20 nM. These responses were corroborated by HPLC analyses on the same extractions and illustrate the applicability of electroanalysis as an analytical technique.

The mycotoxin zearalenone is known for its harmful effects on livestock reproduction. Animal exposure occurs through feed sources colonized by Fusarium species which produce the mycotoxin. Since regular screening procedures for zearalenone are not conducted on Western Canadian barley, a survey was carried out to test for possible significant levels of contamination. All samples were found to be negative at a detection level of 500 ppb; therefore, feeds formulated from the barley samples sources would not likely cause zearalenone toxicosis problems in livestock. Also, an ELISA method, Agri-Screen™, developed by Neogen Corporation (Lansing, Michigan) was tested and found to be a simple and economical method for pre-screening of feed samples in the field. To study direct toxicological effects of zearalenone on in vitro murine blastocyst development, murine embryos were cultured in medium (Ham's F-10 + estrous cow serum) containing various levels of the mycotoxin. The critical concentration range for zearalenone to cause detrimental effects on blastocyst development was determined to be between 70-160 μg/ml medium. Additionally, a concentration effect on the length of time required to exert deleterious actions was demonstrated. At mycotoxin concentrations of 500 μg/ml medium and above, blastocysts degenerated after 6 h of culture. At a lower concentration level of 160 μg/ml, blastocysts were not affected until 28 h of culture. In order to investigate the direct toxicological effects of zearalenone on in vitro porcine pre-implantation embryo development, attempts were made to develop a successful culture system. Since a suitable system was not developed, toxicological studies were not possible. Possibly, steps in the recovery process could have resulted in detrimental effects before the embryos were placed in culture. Alternatively, the media chosen (Ham's F-10 + estrous cow serum; Minimum essential medium + fetal calf serum) may not be suitable for in vitro culture of porcine pre-implantation embryos. Finally, at a zearalenone concentration level (250 μg/ml medium) found to cause degeneration of murine blastocysts, the in vitro maturation of bovine oocytes in Tissue Culture Medium 199 was not affected. It was suggested that the surrounding cumulus layer acts as a barrier to prevent the mycotoxin from directly acting on the oocyte. / Land and Food Systems, Faculty of / Graduate

M.F. Dutton