Made available in DSpace on 2016-05-02T13:55:30Z (GMT). No. of bitstreams: 1 PolianadeOliveiraCoelho-Dissertacao.pdf: 591748 bytes, checksum: 0ee9a76f737829a2f58b8fd626dbacca (MD5) Previous issue date: 2010-09-17 / Aflatoxins are hepatotoxic, immunosuppressive, mutagenic, carcinogenic and teratogenic metabolites produced by Aspergillus flavus and Aspergillus parasiticus. They are found in various kinds of food, especially grains, and are widely used in the manufacture of animal feed. Aflatoxin B1 has the highest toxic power, and its most important metabolite, aflatoxin M1, is eliminated mainly via the milk. The limit values of aflatoxin accepted by Brazilian legislation are 50 ppb for animal feed, and 0.5 µg/L and 5.0 µg/L, for liquid and powdered milk, respectively. Young people are great milk consumers, and thereby milk toxicity becomes an important public health issue. This study evaluated the fungal microbiota and incidence of aflatoxin M1 in the milk of goats raised in three farms, located in the south of Minas Gerais and Mogiana Paulista region. The culture medium Rose-Bengal Sabouraud agar was used to evaluate food fungal microbiota. The presence of aflatoxin B1 was assessed by thin layer chromatography. Aflatoxin M1 in milk was determined by immunoaffinity column cleanout with high performance liquid chromatography (HPLC). In addition, meteorological conditions were surveyed in the regions studied to measure the pluviometric index, the average temperature and relative humidity of air, where the periods of higher incidence of fungi and toxins were found in feed and milk. Two feed samples were collected: concentrate and fodder, with four replications, in two different seasons (rains and drought). Milk samples were also collected, with four replications. Two experiments were conducted: one in the rainy season; another in drought. Their statistical design was completely randomized and factorial . The Tukey test was applied at the 5% significance level. The results showed that the improved methods were efficient to detect aflatoxin: thin layer chromatography, in feed; and high performance liquid chromatography, in milk. The good quality of the goat milk was confirmed by the absence of aflatoxin in the samples. However, the qualitative analysis of Aspergillus cultures revealed that of the 64 samples analyzed, 22,6% were contaminated by aflatoxin B1. / As aflatoxinas são metabólitos tóxicos produzidos por Aspergillus flavus e Aspergillus parasiticus com efeitos hepatotóxicos, imunossupressores, mutagênicos, carcinogênicos e teratogênicos. Estão presentes em vários alimentos, principalmente nos grãos, sendo amplamente utilizados na fabricação de rações animais. A aflatoxina B1 é a de maior poder tóxico e o seu metabólito é eliminado principalmente no leite, sendo o de maior importância a aflatoxina M1. Os limites aceitos pela legislação brasileira são de 50 ppb de aflatoxinas para alimentos destinados a animais e de 0,5 µg/L e 5,0 µg/L de aflatoxina M1 para leite fluido e em pó, respectivamente. Devido à sua toxicidade, principalmente em indivíduos mais jovens, que são os maiores consumidores de leite, tornam-se um importante problema de saúde pública. Os objetivos deste trabalho foram avaliar a microbiota fúngica e a incidência de aflatoxina B1 em alimentos consumidos por cabras e a incidência de aflatoxina M1 em leite de cabras criadas em 3 propriedades localizadas na região do Sul de Minas Gerais e Média Mogiana Paulista. Os alimentos foram avaliados quanto à microbiota fúngica utilizando-se Ágar Sabouraud Rosa de Bengala. A detecção de aflatoxinas B1 foi avaliada pela técnica de cromatografia em camada delgada, enquanto, no leite, primeiramente foi feita a extração e purificação em colunas de imunoafinidade, sendo a detecção de aflatoxina M1 avaliada utilizando-se a cromatografia líquida de alta eficiência (HPLC). Aliado a estes levantamentos, foi realizada uma análise das condições meteorológicas das regiões de Minas Gerais e São Paulo, aferindo-se o índice pluviométrico, a temperatura média e a umidade relativa do ar onde foram verificadas e comparadas as épocas de maior incidência de fungos e suas toxinas nos alimentos e no leite. Foram colhidas amostras de dois alimentos (concentrado e volumoso) com quatro repetições e em duas épocas diferentes (chuva e seca) e amostras de leite também com quatro repetições em cada época. O delineamento estatístico foi inteiramente casualizado (DIC) em esquema fatorial e foi utilizado o teste Tukey com 5% de nível de significância. Os resultados obtidos revelaram que a otimização das referidas metodologias de cromatografia em camada delgada para análise de aflatoxina em rações e de HPLC no leite caprino mostraram-se eficientes. Em todas as amostras de leite caprino, não foi detectada a presença de aflatoxinas, demonstrando a boa qualidade do leite quanto à contaminação por essas toxinas. Porém, após a análise qualitativa da toxigenicidade das culturas da espécie do gênero Aspergillus, observou-se que, das 64 amostras de rações analisadas, 22,6 % encontravam-se contaminadas com aflatoxina B1.

A aflatoxina M1 (AFM1) é um metabólito hidroxilado da aflatoxina B1 (AFB1), e é detectada no leite, após a ingestão de alimentos contaminados com a AFB1. Foram avaliadas durante o período de dez meses (julho/2009 a abril/2010): a ocorrência de AFM1 em leite bovino cru, produzido em 45 fazendas de três mesorregiões produtoras de leite do Estado de São Paulo (Araçatuba, Bauru e Vale do Paraíba); a variação da contaminação entre as mesorregiões produtoras; e a correlação da concentração de AFM1 com parâmetros de qualidade (contagem bacteriana total e contagem de células somáticas) e composição (teores de gordura, proteína, lactose e sólidos totais). A análise de AFM1 foi realizada empregando-se coluna de imunoafinidade para purificação associada à cromatografia líquida de alta eficiência, com detector de fluorescência. Os valores do limite de detecção e de quantificação foram 0,003 µg L-1 e 0,01 µg L-1, respectivamente. A recuperação média da metodologia avaliada com amostras artificialmente contaminada em três concentrações (0,04, 0,1 e 0,2 µg L-1) foi 83%. A AFM1 foi detectada em 210 (49%) das 429 amostras analisadas numa faixa de concentração de traços a 0,617 µg L-1 com média de 0,017 µg L-1 e mediana igual a não detectada (LD<0,003 µg L-1). A concentração média de AFM1 das amostras da mesorregião de Bauru (0,031 µg L-1) foi estatisticamente maior do que da mesorregião de Araçatuba (0,015 µg L-1) e do Vale do Paraíba (traços). Apenas 3 amostras (0,7%) encontraram-se acima do limite máximo permitido no Brasil (0,5 µg L-1) e 28 amostras (6,5%) apresentaram contaminação acima do limite da Comunidade Européia (0,05 µg L-1). A média dos valores de contagem de células somáticas e de contagem bacteriana total foi de 497 103 células mL-1 e 515 103 UFC mL-1, com faixa de 72 a 1411 103 células mL-1 e 17 a 5014 103 UFC mL-1, respectivamente. Os valores médios dos parâmetros de composição do leite foram: gordura 3,59%, proteína 3,15%, lactose 4,50% e sólidos totais 12,22%. Não foi observada correlação significativa entre a ocorrência de AFM1 e os parâmetros de qualidade e de composição do leite. / The AFM1 is a hydroxylated metabolite of aflatoxin B1 (AFB1), and is detected in milk, after ingestion of food/feed contaminated with B1. During the period of ten months (July/2009 to April/2010), it was evaluated: the occurrence of aflatoxin M1 (AFM1) in raw cow milk produced in 45 farms of three mesoregions of São Paulo State (Araçatuba, Bauru and Vale do Paraíba); the variability of contamination among mesoregions; and the correlation of AFM1 contamination with quality parameters (total bacterial count and somatic cell count) and composition (fat, protein, lactose and total solid contents) of raw milk. The AFM1 analysis were performed using an immunoaffinity column for clean up, and high performance liquid chromatography, with fluorescence detection. The detection and quantification limits were 0.003 µg L-1 and 0.01 µg L-1, respectively. The mean recovery of the methodology obtained with spiked aflatoxin-free milk samples at three concentrations (0.04, 0.1 and 0.2 µg L-1) was 83%. The AFM1 was detected in 210 (49%) of 429 samples ranged from traces to 0.617 µg L-1 with a mean level of 0.017 µg L-1 and median not detected (LOD <0.003 µg L-1). The mean AFM1 concentration of samples from Bauru mesoregion (0.031 µg L-1) was statistically higher than Araçatuba (0.015 µg L-1) and Vale do Paraíba (traces) mesorregions. Only 3 samples (0.7%) were above the maximum limit allowed in Brazil (0.5 µg L-1) and 28 samples (6.5%) were above the limit of the European Community (0.05 µg L-1). The mean values of somatic cell count and total bacterial count was 497 103 cells mL-1 and 515 103 CFU mL-1 ranged from 72-1411 103 cells mL-1 and from 17-5014 103 CFU mL-1, respectively. The mean values of the parameters of milk composition were: fat 3.59%, protein 3.15%, lactose 4.50% and total solids 12.22%. There was no significant correlation among the occurrence of aflatoxin M1 and the quality and composition parameters of milk.

This diploma thesis deals with the development and validation of an analytical method using high performance liquid chromatography method with fluorescence detection for the simultaneous determination of aflatoxins and ochratoxin A in herbal and fruit tea. The theoretical part describes the most common groups of mycotoxins and the most important methods for their determination in food. The great attention is devoted to HPLC method and the overview of the derivatization techniques for aflatoxins B1 and G1 fluorescence response enhancement. The practical part of this study focuses on the optimization of sample extraction and purification, the settings of the instrumental analysis and the photochemical reactor assembly. The thesis also involves the determination of the basic performance characteristics for the successful method validation.

<p>Aflatoxins, secondary metabolites produced by the molds <i>Aspergilllus flavus</i> and<i> A. parasiticus</i>, are estimated to affect upwards of 25% of the world’s global food supply. For Low and Middle-Income Countries like Kenya, a combination of trade, economic, and health challenges related to aflatoxin contamination present a serious threat to food and national security. One option for reducing aflatoxin risks in countries like Kenya is deploying small-scale, reprocessing technologies that degrade aflatoxin in contaminated food products. One potential technology for reprocessing is small-scale extrusion (60 pph) like the TechnoChem Mini-Extruder™.</p><p> First, to understand the extent of aflatoxin contamination in Kenyan maize, two field work trials were conducted in Uasin Gishu County, Kenya. Aflatoxin levels from each sample were analyzed and compared to a variety of agro-economic variables (e.g. farm size) using a stepwise multiple linear regression. Upon analysis, only 5% of maize samples collected during field work tested positive for unsafe levels of aflatoxin ( >10 ppb). Thus, the resulting regression model is highly biased towards predicting low aflatoxin levels. Such bias makes any inferences to predict high aflatoxin levels in maize largely inconclusive. The inherent heterogeneity of aflatoxin and the history of wide-spread contamination in Kenya further supports the conclusion that more studies are needed to understand the true extent of aflatoxin contamination in Uasin Gishu maize.</p><p> Second, to test the effectiveness of small-scale extrusion on aflatoxin degradation in maize, contaminated samples were processed at varying motor frequencies (15, 38, and 50 hz) and moisture contents (35, 40, 45 %wb). Moisture content is significant (p-value < 0.05) in aflatoxin degradation. Total aflatoxin degradation varied between 11 and 83% depending on processing conditions. Maximum degradation occurred at 40 %wb product moisture with a residence time of 265.1 s and an effective shear rate of 56.5 1/s. Thermal degradation is considered negligible due to low temperature increases. Consequently, all degradation is attributed to shear forces inside the extruder. Shear rates were approximated using the Harper model with moisture content and residence time being the most significant factors affecting shear effects on aflatoxin degradation. Although significant aflatoxin degradation occurred in the extruder, further studies are necessary to understand the role of processing parameters on aflatoxin degradation before small-scale extrusion can be confirmed as a viable reprocessing technology.</p>

In this dissertation, an ensemble non-uniform spectral feature selection and a kernel density decision fusion framework are proposed for the classification of hyperspectral data using a support vector machine classifier. Hyperspectral data has more number of bands and they are always highly correlated. To utilize the complete potential, a feature selection step is necessary. In an ensemble situation, there are mainly two challenges: (1) Creating diverse set of classifiers in order to achieve a higher classification accuracy when compared to a single classifier. This can either be achieved by having different classifiers or by having different subsets of features for each classifier in the ensemble. (2) Designing a robust decision fusion stage to fully utilize the decision produced by individual classifiers. This dissertation tests the efficacy of the proposed approach to classify hyperspectral data from different applications. Since these datasets have a small number of training samples with larger number of highly correlated features, conventional feature selection approaches such as random feature selection cannot utilize the variability in the correlation level between bands to achieve diverse subsets for classification. In contrast, the approach proposed in this dissertation utilizes the variability in the correlation between bands by dividing the spectrum into groups and selecting bands from each group according to its size. The intelligent decision fusion proposed in this approach uses the probability density of training classes to produce a final class label. The experimental results demonstrate the validity of the proposed framework that results in improvements in the overall, user, and producer accuracies compared to other state-of-the-art techniques. The experiments demonstrate the ability of the proposed approach to produce more diverse feature selection over conventional approaches.

<p>  This dissertation has two essays that are focused on understanding the effects of technology adoption decisions made by smallholder farmers who have incomplete information. The first essay employed a clustered randomized control trial (RCT) with factorial design in upper Eastern Kenya to estimate the impact of three different interventions at improving credence attributes of smallholder farmers’ maize. This essay also utilized a Becker DeGroot Marschak auction method to determine if farmers were willing to adopt a credence technology, and if yes, if their willingness to pay varied based on having previous experience with this agricultural technology. The second essay used the 2018/19 Ethiopia Socio-economic Survey to analyze the impacts of three sources of measurement error caused by farmers’ misperceptions on maize yields. Moreover, this essay explored how farmers’ incomplete information about adoption of one agricultural input led to misallocation of other complementary inputs. </p>

Peanuts (Arachis-hypogaea) are one of the staples in Ghana, Sub-Saharan Africa, and other developing countries. This leguminous crop is frequently contaminated with aflatoxins, which are secondary metabolites of some Aspergillus fungi, mostly Aspergillus. flavus and Aspergillus parasiticus. Aflatoxins in foods are known to cause liver cancer, stunted growth in children, immune system disorders and economic losses. Aflatoxin contamination of peanuts during storage is worse in the tropics because climatic storage conditions there are almost the same as the optimum conditions for Aspergillus growth: temperature conditions of about 26-43 °C and relative humidity of 62-99%. This study investigated the growth of Aspergillus and the production of aflatoxin in shelled peanuts under varying treatment and packaging conditions. In addition, the appropriate pre-storage treatments and packaging needed to reduce aflatoxin production and to maintain quality of shelled and in-shell peanuts in storage under tropical environments were studied. Another aim was to determine the impact of the switch to hermetic storage on peanut farming and marketing profitability in Ghana. Different peanut treatments, with and without Aspergillus flavus fungi, were packaged in different systems; specifically, polypropylene woven sacks and hermetic packaging. Peanuts were analyzed for fungi growth, aflatoxin production and lipid oxidation (peroxide value and p-Anisidine value). Partial roasting and blanching of peanuts eliminated aflatoxigenic fungi and halted aflatoxin production in stored peanuts, increased the effectiveness of peanut sorting and, hence, helped reduce or eliminate aflatoxin levels along the peanut value chain. Additionally, the results of this study demonstrated that hermetic storage, by suppressing aflatoxin production, has the potential for maintaining peanut quality vis a vis polypropylene woven packaging. Profitability analysis conducted as part of this study revealed that the use of the hermetic storage system would not only improve farmer and trader profits, but also reduce the incidence of various ailments attributed to aflatoxins. / Ph. D.

Stink bugs, including native brown stink bug (Euschistus servus) and invasive brown marmorated stink bug (Halyomorpha halys), cause damage to a variety of crops including field corn (Zea mays). Frequency and size of stink bug infestations have increased in corn fields in the Mid-Atlantic U.S., and there are growing concerns that these infestations may contribute to reductions in grain quality including increased mycotoxin concentrations. Prior research on native and invasive stink bugs has focused on understanding their biology, the damage they cause, and elucidating effective and economic management strategies. However, few studies examined the potential for stink bugs to facilitate fungal infection and mycotoxin contamination of corn grain. Thus, the objectives of this research were to: 1) assess the relationship between invasive brown marmorated stink bug (H. halys) feeding injuries and fumonisin contamination of field corn in the Mid-Atlantic U.S., 2) determine if stink bugs are a vector for mycotoxigenic Fusarium spp. in corn, and 3) evaluate the efficacy of pesticides for mitigating stink bug feeding injury and associated mycotoxin contamination in field corn. A correlation between H. halys feeding injury and fumonisin concentrations was identified, and the ability of H. halys to increase F. verticillioides infection and fumonisin concentrations in corn was demonstrated in field experiments. Fusarium species including fumonisin-producing F. verticillioides and F. proliferatum were isolated from field-collected stink bugs, and in laboratory experiments, E. servus was able to transmit F. verticillioides to non-infected corn ears after feeding on F. verticillioides-infected corn. In field studies, both fungicide and insecticide reduced stink bug-associated mycotoxin concentrations in corn, but levels of control were inconsistent. Thus, additional tactics that target both the stink bug and Fusarium should be implemented to mitigate risks of mycotoxin contamination in corn. / Doctor of Philosophy / Native and invasive stink bugs can severely damage crops including field corn. Frequency and size of stink bug infestations in Mid-Atlantic U.S. corn fields have increased, and there is growing concern that this may contribute to reductions in grain quality. Insect feeding injury is a risk factor for fungal infection and mycotoxin contamination in corn. Mycotoxins are toxic chemicals produced by certain fungi that have detrimental health effects on animals including livestock and humans. The relationship between stink bug feeding injuries and mycotoxin contamination in corn grain is not well understood, and management strategies to minimize the risk of mycotoxin contamination in corn need to be identified. The main goal of this research was to characterize interactions between stink bugs and mycotoxin-producing fungi and identify tactics for controlling both the insect pest and pathogen. Specific objectives were to: 1) assess the relationship between invasive brown marmorated stink bug (H. halys) feeding injuries and fumonisin contamination of field corn in the Mid-Atlantic U.S., 2) determine if stink bugs are a vector for mycotoxin-producing Fusarium spp. in corn, and 3) evaluate the efficacy of pesticides for mitigating stink bug feeding injury and associated mycotoxin contamination in field corn. Results from this work indicated that stink bugs have the ability to cause feeding injuries which facilitate invasion of mycotoxin-producing Fusarium species, leading to increases in mycotoxin concentrations in corn grain. Studies also demonstrated that stink bugs can vector Fusarium species during feeding and increase Fusarium infection of corn resulting in subsequent mycotoxin contamination. Field studies indicated that pesticide applications targeting both the stink bugs and mycotoxigenic fungi may be needed to minimize risk of mycotoxin contamination in corn. However, under low pest pressure, application of pesticides is unlikely to be profitable.

The studies described in this thesis investigated the relationship between DNA repair mechanisms, aflatoxin B1 (AFB1)-induced DNA damage and carcinogenesis. Mice deficient in 8-oxoguanine glycosylase (OGG1, the rate-limiting enzyme in repair of oxidized guanine), mice heterozygous for OGG1, and wild type mice, were exposed to a single tumourigenic dose (50 mg/kg) of AFB1. Neither ogg1 genotype nor AFB1 treatment affected levels of oxidized guanine in lung or liver 2 h post-treatment. ogg1 (-/-) mice had increased susceptibility to AFB1 toxicity, as reflected by increased mortality within one week of AFB1 exposure. AFB1 treatment did not significantly increase lung or liver tumourigenesis compared to DMSO controls. No difference was observed between ogg1 genotypes, although a non-significant trend towards AFB1-treated ogg1 (-/-) mice being more susceptible to tumourigenicity was apparent. Overall, deletion of ogg1 did not significantly affect AFB1-induced DNA damage or tumourigenicity, suggesting that oxidized guanine may not be a major contributor to AFB1-induced tumourigenesis. The effects of AFB1 on DNA repair were assessed in p53 (a protein implicated in regulation of DNA repair) wild type and heterozygous mice. p53 (+/+) mice treated with 0, 0.2 or 1.0 ppm AFB1 for 26 weeks had increased nucleotide excision repair (NER) activities in lung and liver compared to control, which may represent an adaptive response to AFB1-derived DNA adducts. In p53 (+/-) mice, the AFB1-induced increase in NER was significantly attenuated, suggesting that loss of one allele of p53 limits the ability of NER to up-regulate in response to AFB1-induced DNA damage. Twenty-six week exposure to AFB1 did not affect base excision repair (BER) in p53 (+/+) mouse lung or liver compared to control. BER was significantly decreased in livers from mice exposed to 1.0 ppm AFB1 compared to those exposed to 0.2 ppm AFB1, a result that was not due to liver cell death or to altered levels of OGG1 protein. In lungs and livers of p53 (+/-) mice, BER activity was unchanged by AFB1. As such, the difference in BER response between 0.2 ppm and 1.0 ppm AFB1 treatment seen in the p53 (+/+) mice appears to be p53 dependent. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-10-17 22:24:31.577

Dans les pays d’Afrique Sub-Saharienne et d’Asie du Sud-Est, l’infection chronique par le virus de l’hépatite B (VHB) et l’exposition à l’aflatoxine B1 (AFB1) ont un rôle synergique dans le développement du carcinome hépatocellulaire (CHC). Cependant les mécanismes impliqués ne sont pas élucidés à ce jour. Le VHB est un petit virus à ADN qui induit différentes maladies du foie allant du portage asymptomatique au CHC. L’AFB1 est une mycotoxine qui contamine la nourriture. Après activation en époxyde, elle forme des adduits à l’ADN puis des mutations, dont la mutation au codon 249 du gène suppresseur de tumeur TP53 (AGG → AGT, mutation R249S). Nous avons utilisé les caractéristiques uniques de la lignée cellulaire HepaRG pour étudier les interactions entre le VHB et l’AFB1 : ces cellules se différencient in vitro en hépatocytes qui métabolisent l’AFB1 et peuvent être infectés par le VHB. Nous avons montré que l’AFB1 induit une réponse de p53 dose-dépendante et agit comme un agent antiviral en réprimant la production de particules virales après 48h d’exposition. D’autre part, l’infection par le VHB n’a montré aucun effet sur la formation ou la réparation des adduits. De la réparation et de la prolifération cellulaire ont été observées suite au traitement à l’AFB1, suggérant la faisabilité de l’étude de mutations dans ce système, dont R249S. Ces résultats suggèrent que l’AFB1 atténue l’hépatite chronique tout en maintenant les hépatocytes sous intense pression mutagène, ce qui favoriserait la progression vers le CHC / In sub-Saharan Africa and South-East Asia, chronic infection by hepatitis B virus (HBV) and exposure to aflatoxin B1 (AFB1) play a synergic role in the development of hepatocellular carcinoma (HCC). However mechanisms are still largely unknown. HBV is a small DNA virus which induces different liver diseases from asymptomatic carriage to HCC. AFB1 is a mycotoxin which contaminates food. After activation into an epoxide, it forms DNA adducts and mutations, such as R249S mutation at codon 249 in tumor suppressor TP53 gene (AGG → AGT). We have taken advantage of the unique features of the cell line HepaRG to investigate interactions between both risk factors: cells differentiate in vitro into hepatocytes which metabolize AFB1 and can be infected by HBV. We have shown that AFB1 induces a dose-dependent p53 response and act as an antiviral agent by repressing production of HBV particles after 48 hours of exposure. Nevertheless HBV infection had no effect on adduct formation or repair. Moreover DNA synthesis activity associated to DNA repair and cell proliferation were observed following AFB1 treatment, suggesting the feasibility of mutation research in this model, especially R249S. Overall these results suggest that AFB1 may attenuate HBV chronic hepatitis while maintaining hepatocytes under intense mutagenic pressure, thus enhancing the progression towards HCC