Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biological Sciences at Technikon Natal, 2000. / Spice oils are known to exhibit antifungal activity and therefore have the potential to control mycotoxin production. There is a need in the food industry to find measures to control mycotoxins that are frequently associated with grains that form the staple diet of the majority of the population in South Africa. Clove, cinnamon, oregano, tumeric, eucalyptus, neem, aniseed, mace and nutmeg oils were tested to determine their inhibitory potential against growth of Aspergillus parasiticus and Fusarium moniliforme using the agar overlay technique. Varying concentrations of the spice oils, ranging from 0.1 ppm to 2.0 ppm, were incorporated into broth cultures of A. parasiticus and maize patty cultures ofF. moniliforme. Levels of production of aflatoxins and fumonisin were determined using standard thin layer chromatography and highpressure liquid chromatography methods. In addition, the active component of the spice oils were isolated, characterised and tested. The inhibitory potential of these compounds for field use was tested by incorporating clove oil, whole cloves and ground cloves in samp / M

Aflatoxin B1 (AFB1), a potent hepatotoxic and hepatocarcinogenic mycotoxin synthesised by toxigenic fungi (Aspergillus flavus and Aspergillus parasiticus), is a common contaminant of many cereal commodities consequently posing a major threat to human and animal health. Sutherlandia frutescens (SF), a traditional medicinal plant endemic to Southern Africa, is commonly used by many cultures as a tonic for various health-related conditions. Incidentally, the present study aimed at investigating the potential hepatoprotective capacity of SF and L-canavanine (L-can, a major constituent of SF) against AFB1-induced cytotoxicity in human HepG2 cells and used a standard treatment procedure of 24 h. Cell viability was evaluated using the methyl thiazol tetrazolium (MIT) assay, which effectively demonstrated the ability of SF, when administered individually and in combination with AFB1, to be significantly cytotoxic to HepG2 cells in a dose-dependant manner. Reactive oxygen species (ROS) and consequent peroxidative damage caused by AFB1 are considered to be the main mechanisms leading to hepatotoxicity and was confirmed by the thiobarbituric acid reactive substances (TBARS) assay which revealed that AFB1 mediated a significant increase in lipid peroxidation. Additionally, comet assay analysis demonstrated the most pronounced effect to be observed following administration of AFB1. In contrast, AFB1-mediated genotoxicity was significantly reduced by SF and L-can. Such amelioration can be attributed to the marked increases in glutathione (OSH) levels observed after the co-administration of SF and L-can with AFB1. Cytoprotection by SF and L-can against AFB1-induced toxicity was further substantiated by the significant increases in heat shock protein 70 expression. Moreover, when SF and L-can were co-administered along with AFB1, analysis by flow cytometry revealed that AFB1 induced increases in apoptosis and necrosis were reduced. The findings of this study propose that SF and L-can may be selectively effective in alleviating AFB1-induced cytotoxicity and lends pharmacological credibility to the suggested ethnomedical uses of SF. However, the exact mechanism of action and the extracts efficacy in humans requires further authentication. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2008.

A avaliação da capacidade produtora de micotoxinas vem sendo utilizada como uma importante ferramenta na identificação de espécies conhecidamente toxigênicas. Poucos são os métodos rápidos e alternativos disponíveis para a determinação do potencial toxigênico de espécies do gênero Aspergillus. Neste contexto, o objetivo deste trabalho foi avaliar a capacidade produtora de aflatoxina B1, em diferentes condições de cultivo, por três isolados de Aspergillus flavus, produtores de aflatoxina B1. O delineamento experimental baseou-se em um planejamento 2³ completo, tendo como variáveis independentes a temperatura (20-40°C), o tempo de incubação (7-21 dias) e o pH (2,0-6,0) nos meio sintéticos (YES, CYA e Sabouraud). As melhores condições encontradas foram aplicadas em testes com meio natural (arroz) e isolados a principio não-aflatoxigênicos. Aflatoxina B1 foi extraída diretamente dos meios sintéticos com clorofórmio e do arroz com metanol. A identificação e quantificação do composto foi realizada por Cromatografia em Camada Delgada e Fotometria Fotográfica. O meio YES se mostrou o melhor para detecção do potencial toxigênico, seguidos de melhor pH 4,0 e 5,2, e temperatura de 20º e 25ºC e tempo de incubação de 11 e 14 dias. O isolado A43, em temperatura de 25º, pH 5,2 e tempo de incubação de 11 dias, mostrou a maior produção de aflatoxina B1, com 206,05 ng. No arroz, os isolados revelaram produção de aflatoxina, apenas a partir do 14ºdia. Dos 30 isolados a princípio não-aflatoxigênicos testados inicialmente em agar coco, 12 apresentaram resultado positivo nos meios e condições aqui apresentados. / Mycotoxins producing capacity evaluation has being used as an important tool, in the identification of toxigenic species. A few of them are available as alternative rapid methods for the determination of the toxigenic potential of species Aspergillus. The objective of this work was to evaluate the aflatoxin B1 producing capacity in different conditions of culture by three Aspergillus flavus. The experimental delineation was based on a 2³ factorial design. To test the effect of three independent variables, the temperature (20-40°C), the incubation time (7-21 days) and pH (2,0 -6,0) in the synthetic medium (YES, CYA and Sabouraud) were applied in the program STATISCA 7.0. The best joined conditions had been applied in tests with natural medium (rice) and isolated tested as nonaflatoxigenics. Aflatoxin B1 was extracted directly from sintetic mediuns by chloroform and from rice by methanol. Thin-layer chromatography (TLC) and Photometric Photography were the methods utilized for the identification and quantification of aflatoxin B1. YES was the best medium for the detention of toxigenic potential, at pH 4,0 and 5,2, temperature of 20º and 25ºC and incubation time of 11 and 14 days. The isolated A43, at temperature of 25ºC, pH 5,2 and incubation time of 11 days showed the biggest aflatoxin B1 production (206,05 ng). Aflatoxin production in rice occurred only after 14 days. 12 of the 30 non aflatoxigenic isolates showed aflatoxin production in the media and conditions tested.

A avaliação da capacidade produtora de micotoxinas vem sendo utilizada como uma importante ferramenta na identificação de espécies conhecidamente toxigênicas. Poucos são os métodos rápidos e alternativos disponíveis para a determinação do potencial toxigênico de espécies do gênero Aspergillus. Neste contexto, o objetivo deste trabalho foi avaliar a capacidade produtora de aflatoxina B1, em diferentes condições de cultivo, por três isolados de Aspergillus flavus, produtores de aflatoxina B1. O delineamento experimental baseou-se em um planejamento 2³ completo, tendo como variáveis independentes a temperatura (20-40°C), o tempo de incubação (7-21 dias) e o pH (2,0-6,0) nos meio sintéticos (YES, CYA e Sabouraud). As melhores condições encontradas foram aplicadas em testes com meio natural (arroz) e isolados a principio não-aflatoxigênicos. Aflatoxina B1 foi extraída diretamente dos meios sintéticos com clorofórmio e do arroz com metanol. A identificação e quantificação do composto foi realizada por Cromatografia em Camada Delgada e Fotometria Fotográfica. O meio YES se mostrou o melhor para detecção do potencial toxigênico, seguidos de melhor pH 4,0 e 5,2, e temperatura de 20º e 25ºC e tempo de incubação de 11 e 14 dias. O isolado A43, em temperatura de 25º, pH 5,2 e tempo de incubação de 11 dias, mostrou a maior produção de aflatoxina B1, com 206,05 ng. No arroz, os isolados revelaram produção de aflatoxina, apenas a partir do 14ºdia. Dos 30 isolados a princípio não-aflatoxigênicos testados inicialmente em agar coco, 12 apresentaram resultado positivo nos meios e condições aqui apresentados. / Mycotoxins producing capacity evaluation has being used as an important tool, in the identification of toxigenic species. A few of them are available as alternative rapid methods for the determination of the toxigenic potential of species Aspergillus. The objective of this work was to evaluate the aflatoxin B1 producing capacity in different conditions of culture by three Aspergillus flavus. The experimental delineation was based on a 2³ factorial design. To test the effect of three independent variables, the temperature (20-40°C), the incubation time (7-21 days) and pH (2,0 -6,0) in the synthetic medium (YES, CYA and Sabouraud) were applied in the program STATISCA 7.0. The best joined conditions had been applied in tests with natural medium (rice) and isolated tested as nonaflatoxigenics. Aflatoxin B1 was extracted directly from sintetic mediuns by chloroform and from rice by methanol. Thin-layer chromatography (TLC) and Photometric Photography were the methods utilized for the identification and quantification of aflatoxin B1. YES was the best medium for the detention of toxigenic potential, at pH 4,0 and 5,2, temperature of 20º and 25ºC and incubation time of 11 and 14 days. The isolated A43, at temperature of 25ºC, pH 5,2 and incubation time of 11 days showed the biggest aflatoxin B1 production (206,05 ng). Aflatoxin production in rice occurred only after 14 days. 12 of the 30 non aflatoxigenic isolates showed aflatoxin production in the media and conditions tested.

A avaliação da capacidade produtora de micotoxinas vem sendo utilizada como uma importante ferramenta na identificação de espécies conhecidamente toxigênicas. Poucos são os métodos rápidos e alternativos disponíveis para a determinação do potencial toxigênico de espécies do gênero Aspergillus. Neste contexto, o objetivo deste trabalho foi avaliar a capacidade produtora de aflatoxina B1, em diferentes condições de cultivo, por três isolados de Aspergillus flavus, produtores de aflatoxina B1. O delineamento experimental baseou-se em um planejamento 2³ completo, tendo como variáveis independentes a temperatura (20-40°C), o tempo de incubação (7-21 dias) e o pH (2,0-6,0) nos meio sintéticos (YES, CYA e Sabouraud). As melhores condições encontradas foram aplicadas em testes com meio natural (arroz) e isolados a principio não-aflatoxigênicos. Aflatoxina B1 foi extraída diretamente dos meios sintéticos com clorofórmio e do arroz com metanol. A identificação e quantificação do composto foi realizada por Cromatografia em Camada Delgada e Fotometria Fotográfica. O meio YES se mostrou o melhor para detecção do potencial toxigênico, seguidos de melhor pH 4,0 e 5,2, e temperatura de 20º e 25ºC e tempo de incubação de 11 e 14 dias. O isolado A43, em temperatura de 25º, pH 5,2 e tempo de incubação de 11 dias, mostrou a maior produção de aflatoxina B1, com 206,05 ng. No arroz, os isolados revelaram produção de aflatoxina, apenas a partir do 14ºdia. Dos 30 isolados a princípio não-aflatoxigênicos testados inicialmente em agar coco, 12 apresentaram resultado positivo nos meios e condições aqui apresentados. / Mycotoxins producing capacity evaluation has being used as an important tool, in the identification of toxigenic species. A few of them are available as alternative rapid methods for the determination of the toxigenic potential of species Aspergillus. The objective of this work was to evaluate the aflatoxin B1 producing capacity in different conditions of culture by three Aspergillus flavus. The experimental delineation was based on a 2³ factorial design. To test the effect of three independent variables, the temperature (20-40°C), the incubation time (7-21 days) and pH (2,0 -6,0) in the synthetic medium (YES, CYA and Sabouraud) were applied in the program STATISCA 7.0. The best joined conditions had been applied in tests with natural medium (rice) and isolated tested as nonaflatoxigenics. Aflatoxin B1 was extracted directly from sintetic mediuns by chloroform and from rice by methanol. Thin-layer chromatography (TLC) and Photometric Photography were the methods utilized for the identification and quantification of aflatoxin B1. YES was the best medium for the detention of toxigenic potential, at pH 4,0 and 5,2, temperature of 20º and 25ºC and incubation time of 11 and 14 days. The isolated A43, at temperature of 25ºC, pH 5,2 and incubation time of 11 days showed the biggest aflatoxin B1 production (206,05 ng). Aflatoxin production in rice occurred only after 14 days. 12 of the 30 non aflatoxigenic isolates showed aflatoxin production in the media and conditions tested.

D.Tech. (Biomedical technology) / The majority of the South African rural black population remain is exposed to HIV/ AIDS and other chronic diseases, tuberculosis, malaria and cancer. The effect of single and combined mycotoxins on their health and particularly their immune system is unknown and remain of concern as these populations are on daily basis exposed more than one mycotoxin at once. The aim of this study was to evaluate the exposure of South African rural black populations to mycotoxins via animal products in comparison to urban populations and to assess the effect of the major mycotoxins (fumonisin B1 (FB1), aflatoxin B1 (AFB1) and ochratoxin A (OTA)) mostly present in their food on human and animals (pigs) mononuclear cells and by extrapolation, evaluate possibilities of these mycotoxins on the immune system. To achieve this, animal feed and animal products (milk, serum, and tissues) obtained from selected rural and commercial farms in selected areas of South Africa were analysed for fungal and mycotoxins contamination. It was found in this study that almost all of the samples from both areas were contaminated with the major mycotoxin producing fungal strains (Fusarium, Aspergillus and Penicillium spp.) with the most prominent among them being Aspergillus flavus (87%), A. parasiticus (43%), A. niger (69%), A. ochraceus (42%), A. candidus (23%), F. verticillioides (98%) F. graminearum (67%) and P. Verrucosum (48.9%) and in commercial samples A. flavus (98%), A. parasiticus (51%), A. ochraceus (65%), A. niger (31%), A. candidus (21%), F. verticillioides (F. moniliforme) (68%), F. graminearum (43%) and P. verrucosum (7%). While, the three main mycotoxins were also present and contaminated most samples with fumonisins (FBs) 0in rural and commercial samples at 90.6% and 93.3% respectively with respective means values of 10136.4 ppb and 1045.4 ppb. Aflatoxins (AFs) contamination was of 92.0% in rural samples and 96.2% in commercial samples with means concentrations of 168.8 ppb and 294.1 ppb respectively. While 85.4% and 83.7% of rural and commercial samples respectively were contaminated with ochratoxin A (OTA), with mean concentrations of 67.6 ppb and 89.4 ppb respectively. Zearalenone (ZEA) concentrations were of 43.6 ppb in rural samples and 62.7 ppb in commercial samples with respective contamination of 50.6% and 55.3%. In addition, a co-occurrence of fungi and mycotoxins contaminations was found in both rural and commercial samples. It was found that, 50.5% of rural and 53% of commercial samples were contaminated with all four analyzed mycotoxins. (FBs, AFs, OTA and ZEA), whereas, 81.2% and 79.5% of samples respectively from rural and commercial farms were contaminated with FBs, AFs and OTA mycotoxins simultaneously. The above-obtained results are of significance in this study as they confirm the hypothesis of fungal contamination and mycotoxin co-occurrence in South African feed and their possible combined effects on consumers.

M.Sc. / A phytotoxic protein-lipopolysaccharide complex (PLPC) was isolated from 7 day old culture filtrates of Verticillium dahliae. The complex was purified to electrophoretic homogeneily by means of acetone precipitation, gel, chromatography and preparative agarose electrophoresis with a yield of 4.5 mg PLPC per litre culture filtrate ...

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biological sciences at the ML Sultan Technikon, 2000. / Plant medicine is an important area of commercial activity in South Africa. This is a rapidly expanding market, thus we are evaluating natural and stressinduced compounds (phytoalexins) from plants as agents that may be able to control mycotoxins. Natural compounds from Bridelia micrantha, Warburgia salutaris, Lippia javanica and Scenecio serratuloides and stress-induced compounds (phytoalexins) from Citrus sinensis cv Valencia were screened for antitunqal and antimycotoxic activity by bioautography against a test organism (Cladosporium cladosporoides) and mycotoxin producing fungi (Fusarium moniliforme and Aspergillus flavus). / M

O objetivo deste estudo foi verificar a ocorrência de aflatoxina e fumonisina no sistema de produção de frango de corte e o impacto destas micotoxinas nos índices produtivos em uma empresa integradora localizada no Estado de São Paulo. Adicionalmente, foram identificados os principais fatores para a produção de micotoxinas em rações e a ocorrência de resíduos de aflatoxinas e fumonisinas em tecidos comestíveis de frango (músculo peitoral, fígado e moela). Foram realizadas as contagens de fungos e leveduras totais, de fungos dos gêneros Aspergillus ssp. e Fusarium ssp. e quantificação de aflatoxina e fumonisina nas principais matérias-primas da ração (milho e farelo de soja), na ração de abate e na cama de frango. O isolamento de fungos nas amostras de milho, farelo de soja e ração foi realizado em ágar DG18, enquanto que, para as amostras de cama de frango, utilizou-se o ágar PDA. Para a extração de aflatoxinas e fumonisinas, foram utilizadas colunas de imunoafinidade (Neogen®) e colunas SAX de troca iônica, respectivamente. A quantificação das aflatoxinas e fumonisinas foi realizada através de cromatografia líquida de alta eficiência (CLAE). O milho foi o alimento onde foi observada a maior frequência de Aspergillus ssp. e Fusarium ssp., e também maior positividade para aflatoxinas e fumonisinas, sendo que uma das amostras ultrapassou o limite de aflatoxinas recomendado pelo Ministério da Agricultura Pecuária e Abastecimento (MAPA). As quantidades de aflatoxinas e fumonisinas encontradas na ração não influenciaram significativamente os índices produtivos. Não foram encontrados níveis detectáveis de resíduos de aflatoxinas e fumonisinas nos tecidos analisados. Embora não tenham sido observadas lesões macroscópicas no fígado e bursa das aves, foram constatadas alterações histopatológicas nessas vísceras, as quais são compatíveis com lesões causadas pela ingestão de aflatoxinas e fumonisinas. / The objective of this study was to verify the occurrence of aflatoxin and fumonisin in poultry feed and their influence on poultry productivity at company located in São Paulo State. Supplementary, were identified the main factors that cause mycotoxin production in poultry feed and determine the occurrence of aflatoxins and fumonisins residues in edible parts of poultry (breast, liver and gizzard). The total mold and yeast counting of Aspergillus ssp. and Fusarium ssp. genus and quantification of aflatoxin and fumonisin were determined in the main feed ingredients (corn and soybean meal), in finishing diets and bedding. The fungi from corn, soybean meal and feed were isolated in DG18 agar, whereas, the fungi from bedding was used PDA agar. Aflatoxins and fumonisins, were extracted using an immunoaffinity column (Neogen®) and a SAX column, respectively. Aflatoxins and fumonisins were quantified by high performance liquid chromatografy (HPLC). The corn showed the highest frequency of Aspergillus ssp. and Fusarium ssp. and also the highest positivity for aflatoxins and fumonisins, there was one corn sample that exceeded the recommendations of the Brazilian Ministry of Agriculture. The levels of aflatoxins and fumonisins in the feed did not significantly influence productivity. There were not detectable levels of aflatoxins and fumonisins on analysed tissues. Although macroscopic lesions were not observed in liver and bursa, histopathological changes were observed in these organs, which are consistent with injuries caused by the aflatoxin and fumonisin consumption.

Foi pesquisada a habilidade de carunchos Sitophilus zeamais em veicular esporos de Aspergillus flavus e Fusarium verticillioides e a conseqüente produção de micotoxinas. Grãos de milho foram mantidos em frascos conectados por uma mangueira formando um sistema fechado (lados A e B) e divididos em seis grupos: G1 (milho + caruncho - Lado A); G2 (Milho + A. flavus - Lado A); G3 (milho + A. flavus + caruncho - Lado A); G4 (milho + F. verticillioides - Lado A), G5 (milho + F. verticillioides + caruncho - Lado A) e G6 (milho + A. flavus + F. verticillioides + caruncho - Lado A). O lado B continha grãos estéreis. Após 10, 20 e 30 dias de incubação foram realizadas: pesagem, atividade de água, microbiota fúngica, determinação de micotoxinas, análise nutricional, microscopia eletrônica de varredura e PCR-RT em tempo real. Frente aos resultados obtidos constata-se a importância do Sitophillus zeamais como vetor de fungos e a importância de boas práticas de manipulação e armazenamento de grãos, visando reduzir os riscos de contaminação e deterioração. / The weevils Sitophilus zeamais ability was examined to propagate spores of Aspergillus flavus and Fusarium verticillioides and the production of mycotoxins. Corn grains was conserved in flasks connected by a rubber to form a closed system (side A and B) and divided in six groups: G1 (corn + weevil - side A); G2 (corn + A. flavus - side A); G3 (corn + A. flavus + weevil - side A); G4 (corn + F. verticillioides - side A), G5 (corn + F. verticillioides + weevil - side A) e G6 (corn + A. flavus + F. verticillioides + weevil - side A). The side B contained sterile grains. After 10, 20 and 30 days of incubation were realized: weighing, activity water, mycoflora, determination of mycotoxins, nutritional analysis, scanning electron microscope and Real time PCR-RT. In front of the results was observed the importance of Sitophilus zeamais like a fungus vector and the importance of Good Manufacturing Practices and Stores of grains, to reduce the risks of contamination and deterioration.