Mycotoxins have become an important issue for the grain industry and animal producers with a growing interest in the decontamination and remediation of highly contaminated feedstuffs. Practical methods to detoxify mycotoxin-contaminated grain on a large scale and in a cost-effective manner are essential but not currently available. The most recent and promising approach to detoxify mycotoxin-contaminated grain is the use of non-nutritive adsorbents, which bind the aflatoxin and thereby reduce their absorption from the gastrointestinal tract. Humic acids are products of chemical and biological transformations of animal and plant residues and are widely distributed in nature. Humic acids have some therapeutic characteristics and a strong binding affinity for several compounds. A South African company developed an effective large-scale regeneration process for humic acids from coal, called oxihumate. This study evaluated the effectiveness of oxihumate to adsorb mycotoxins, for the purpose of developing it as a commercial mycotoxin binder to be used in the preventative management of contaminated poultry feedstuffs. The in vitro affinity and adsorption capacity of oxihumate to aflatoxin was evaluated and the efficacy of oxihumate as an aflatoxin binder in broiler feeds in vivo was determined. The data showed adsorptions of about 10.3, 7.4 and 11.9 mg aflatoxin B1/g oxihumate at pH 3, 5 and 7, respectively. Oxihumate adsorbed 1.2, 2.6 and 8.5 mg aflatoxin G2/g at pH 3, 5 and 7, respectively. Oxihumate supplementation at a concentration of 3.5 g/kg feed was effective in diminishing the growth inhibitory effects of aflatoxin and apparent protection was noted for some of the organ, haematological and serum biochemical changes associated with aflatoxicosis. These results suggest that oxihumate could alleviate some of the toxic effects of aflatoxin in growing broilers, and when used with other sound mycotoxin management practices, might prove beneficial in the preventative management of aflatoxin-contaminated feedstuffs for poultry. The improvement observed during this specific study was, however, not satisfactory enough to recommend oxihumate as a commercially available product. / Thesis (PhD (Animal and Wildlife Sciences))--University of Pretoria, 2007. / Animal and Wildlife Sciences / unrestricted

O projeto teve por finalidade isolar e identificar fungos, avaliar o potencial toxigênico dos isolados de Aspergillus spp. e Fusarium spp., detectar e quantificar aflatoxinas B1, B2, G1, G2 e fumonisinas B1 e B2 em amostras de misturas de cereais. Foram analisadas 15 marcas de misturas de cereais, prontas para o consumo, adquiridas de supermercados e de empresas que comercializam o produto nacionalmente via internet. Foram adquiridas amostras por sete meses, totalizando 105 amostras ao final do experimento. A contagem de bolores nas amostras variou de 1,0 x 101 a 2 x 105 unidades formadoras de colônias (UFC)/g, com isolamento de sete cepas de Aspergillus flavus. As aflatoxinas B1 e G1 foram detectadas em poucas amostras e em baixos níveis, sendo que estes resultados podem ser devidos à baixa atividade de água nos produtos avaliados, a qual foi inferior a 0,63. A fumonisina B1 foi detectada em 84,8% das amostras, no entanto, a ingestão diária provável calculada para as fumonisinas esteve abaixo da recomendação do JECFA. Apenas uma amostra apresentou níveis de fumonisinas acima do limite esperado para 2016. Adicionalmente, foi observado que 21% das amostras apresentaram mais de um tipo de micotoxina, o que poderia conduzir à potencialização de efeitos tóxicos. / The project aimed to isolate and identify fungi, evaluate the toxigenic potential of isolates of Aspergillus spp. and Fusarium spp. detect and quantify aflatoxins B1, B2, G1, G2 and fumonisins B1 and B2 in samples of cereal mixtures. We analyzed 15 brands of cereal mixtures ready to eat adding up to 105 samples at the end of the experiment. Samples were acquired in supermarkets and from companies that market the product nationally by internet. The mold count in the samples ranged from 1.0 x 101 to 2 × 105 colonies forming units (CFU)/ g, with isolation of seven strains of Aspergillus flavus. Aflatoxin B1 and G1 were detected in a few samples and at low levels, what might be due to the low water activity in the product reviews, which was less than 0.63. Fumonisin B1 was detected in 84.8% of the samples, however, the probable daily intake calculated for fumonisin was bellow the JECFA recommendation. Only one sample showed fumonisin levels above the expected limit for 2016. Additionally, it was observed that 21% of the samples presented more than one type of mycotoxin, which could lead to enhancement of toxic effects.

Espécie típica da floresta amazônica, a castanha-do-brasil (Bertholletia excelsa, H.B.K.) é uma commodity selvagem, que se destaca na economia regional por ser um produto de alto valor econômico nos mercados nacional e internacional. Apresenta também reconhecido valor nutricional, decorrente de sua composição em lipídeos, proteínas e vitaminas, além de constituir uma excelente fonte de selênio. Entretanto, as condições climáticas em que esta cultura se insere, o baixo nível tecnológico característico de sua cadeia produtiva e as condições inadequadas de manejo e manuseio favorecem a contaminação por aflatoxinas, como também, a deterioração por oxidação por ser um alimento constituído de 60 a 70% de lipídeos. Estas deficiências no processo de extrativismo geram problemas para indústria processadora, que deve fornecer ao consumidor um produto de qualidade nutricional e sanitária. Este estudo objetivou caracterizar a castanha-do-brasil em diferentes etapas do processamento, como também avaliar a sua qualidade ao longo do beneficiamento. Foram analisadas amostras coletadas em 3 etapas, ao longo do processamento industrial, representando o ínicio do processamento (castanha in natura), fase intermediária (castanha dry) e fase final (amêndoas desidratadas). Em cada etapa foram realizadas 8 repetições de amostragens. A composição nutricional foi mantida ao longo do processamento, com predominância do ácido graxo linolênico (ômega-6) nas amostras. O índice de acidez da fração lipídica apresentou resultados satisfatórios, que se encaixam dentro dos limites da legislação vigente para óleos brutos extraídos a frio, com 2,20±0,84, 2,48±0,97 e 0,18±0,07 mg KOH/g de óleo para a castanha in natura, dry e amêndoa desidratada respectivamente. O índice de peróxido da fração lipídica nas amostras in natura apresentaram média de 0,018±0,010 mmol/kg, dry 0,032±0,011mmol/kg e na amêndoa desidratada 0,044±0,011 mmol/kg. Mesmo com o aumento do teor de hidroperóxidos em função do processamento, todas as amostras estão dentro dos padrões estabelecidos na legislação brasileira. Os resultados para absorbância em luz ultravioleta a 232 nm corroboram os resultados da formação de peróxidos, com diferença significativa entre as amostras. Os valores apresentados pelas castanhas in natura, dry e amêndoa desidratada foram de 2,14±0,28, 1,60±0,47 e 3,08±0,37 E1% 1cm, respectivamente. Apesar das diferenças entre indicadores do estado oxidativo das amostras, não houve diferença significativa na estabilidade oxidativa da fração lipídica, quantificada pelo período de indução em Rancimat, cuja média foi de 4,29 horas. A composição em ácidos graxos das amostras também foi similar, sem diferenças significativas entre grupo de ácidos graxos saturados, monoinsaturados e polinsaturados. Em todas as amostras os resultados para coliformes em termos de E. coli foram menores que 1 NMP/g, e ausência de Salmonella em 25 g de castanha. Entretanto, elevados índices de contaminação por aflatoxinas foram encontrados. Apenas 5 das 24 amostras estavam dentro dos limites estabelecidos nas legislações brasileiras e da União Europeia. Contudo, a presença de contaminação por aflatoxinas nas amostras de amêndoas desidratadas, foi reduzida significativamente, após as etapas de seleção e classificação das castanhas. Por meio destes resultados concluímos que as etapas do processamento interferem positivamente na qualidade da castanha-do-brasil. / Typical specie of the Amazon rainforest, the Brazil nut (Bertholletia excelsa, H.B.K.) is a wild commodity that stands out in the regional economy as a product of high economic value both in Brazilian and international markets. It presents also a recognized nutritional value, due to its composition in lipids, proteins and vitamins, in addition to being an excellent source of selenium. However, the climatic conditions of production regions, the low technological level of its supply chain and the unsuitable conditions of harvesting and handling favor the aflatoxins contamination with consequent risk to consumer health and economic losses, as well as, the deterioration by oxidation because it is a food consisting of 60 to 70% lipids. The deficiencies in processes create problems for the processing industry, that should provide the consumer one product with nutritional and health quality. This study aimed to characterize the Brazil nut at different stages of processing, but also assess their quality along the processing. Samples were analyzed in 3 stages, colected at each step of industrial processing, representing the beginning of processing (in nature), intermediate stage (dry nut) and final product (dehydrated kernel). The nutritional composition was maintained throughout the process, with predominant linolenic acid (omega-6) in cold pressed oil samples. Lipidic fraction samples presented low acid values, showing satisfactory results that fit within the limits of current legislation for crude oils: 2.20±0.84, 2.48±0.97 and 0.18±0.07 mg KOH/g in in nature, dry and dehydrated kernel samples, respectively. The peroxide value of the lipid fraction in fresh samples was 0.018±0.010 mmol/kg, in dry sample 0.032±0.011 mmol/kg, and in the dehydrated kernel, 0.044±0.011 mmol/kg. Even with the significant increase in oxidative index, the samples are within the standards established by Brazilian law. The results for absorbance in ultraviolet light (232 nm) corroborate the results of hydroperoxides formation, with significant difference among samples. Absorptivity at 232 nm in in nature nuts, dry and dehydrated kernel were 2.14±0.28, 1.60±0.47 and 3.08±0.37 E1% 1cm, respectively. There was no significant difference in oxidative stability measures by induction period in Rancimat. Samples presented a mean of 4,29 hours and the same fatty acid composition (total saturated, monounsaturated and polunsaturated fatty acids). In all samples the results for coliforms in terms of E. coli were <1 NMP/g, and there were absence of Salmonella in 25 g of nuts. However high levels of contamination by aflatoxins were found. Only 5 of the 24 samples were within the limits established in the Brazilian legislation and the European Union. But, the presence of aflatoxins in dehydrated kernel, was significantly reduced, following the steps of selection and classification of nuts. Through of these results we can conclude that the processing steps positively affect the quality of the Brazil nut.

Chilean red chili peppers contaminated with aflatoxins were reported in a previous study. If the development of gallbladder cancer (GBC) in Chile is associated with a high level of consumption of aflatoxin-contaminated red chili peppers, such peppers from other countries having a high GBC incidence rate may also be contaminated with aflatoxins. We aimed to determine whether this might be the case for red chili peppers from Bolivia and Peru. A total of 7 samples (3 from Bolivia, 4 from Peru) and 3 controls (2 from China, 1 from Japan) were evaluated. Aflatoxins were extracted with acetonitrile:water (9:1, v/v) and eluted through an immuno-affinity column. The concentrations of aflatoxins B1, B2, G1, and G2 were measured using high-performance liquid chromatography (HPLC), and then the detected aflatoxins were identified using HPLC-mass spectrometry. In some but not all of the samples from Bolivia and Peru, aflatoxin B1 or aflatoxins B1 and B2 were detected. In particular, aflatoxin B1 or total aflatoxin concentrations in a Bolivian samples were above the maximum levels for aflatoxins in spices proposed by the European Commission. Red chili peppers from Bolivia and Peru consumed by populations having high GBC incidence rates would appear to be contaminated with aflatoxins. These data suggest the possibility that a high level of consumption of aflatoxin-contaminated red chili peppers is related to the development of GBC, and the association between the two should be confirmed by a case-control study.

Toxigenic Aspergillus species produce mycotoxins that are carcinogenic, hepatotoxic and teratogenic immunosuppressing agents in both human and animals. Kenya frequently experiences outbreaks of aflatoxicosis with the worst occurring in 2004, which resulted in 125 deaths. This study sought to find possible reasons for frequent aflatoxicosis outbreaks in Kenya by isolating Aspergillus flavus strains from maize kernels sampled from different climatic regions of Kenya. Using diagonal transect random sampling, maize kernels were collected from Makueni, Homa Bay, Nandi, and Kisumu regions. The genetic diversity and variation among the isolates was examined by characterising the strains according to morphology, phenotype, vegetative compatible groups and molecular systematics. Selected atoxigenic and aflatoxigenic A. flavus isolates were also further analysed for aflatoxin production potential using quantitative real-time PCR and various bioanalytical techniques. The influence of the maize lines grown in Kisumu, Homa Bay, Nandi and Makueni region on A. flavus infection and aflatoxin production was also examined and served as the basis for an in vitro biocontrol assay. Out of 37 isolates identified, nitrate non-utilizing auxotroph’s complementation test revealed 20 vegetative compatibility groups. These groups were further designated using the prefix ʻʻKVCGʼʼ, where ʻʻKʼʼ represented Kenya and consequently assigned numbers 1 to 20 based on our findings. KVCG14 and KVCG15 had highest distribution frequency (n = 13; 10.8 %). The distribution of the L, S and S/L- morphotypes across the regions were 57 % (n = 21); 7 % (n = 3) and 36 % (n = 13) respectively. The phylogenetic analysis exhibited high diversity of A. flavus isolates from Makueni. ITS1 and ITS2 markers did not reveal significant information within intraspecies speciation of A. flavus. Furthermore, a unique isolate (KSM015) was identified that had characteristics of S-morphotype, but produced both aflatoxins B and G. Coconut agar medium (CAM) assay, TLC, HPLC and LCMS/MS analyses confirmed the presence or absence of aflatoxins in selected toxigenic and atoxigenic isolates. qPCR analysis revealed aflP, aflS, aflR and aflO transcripts as the most upregulated genes across the tested isolates whereas false detection of aflD gene transcript was observed in both induced and uninduced A. flavus isolates. Diversity Index (H) analyses ranged from 0.11 (Nandi samples) to 0.32 (Kisumu samples). Heterokaryon compatibility ranged from 33 % (for the Makueni samples, n = 3) to 67 % (Nandi samples, n = 6). The KDV1 maize line was more sensitive to A. flavus infection in comparison to GAF4. We also tested the biocontrol of atoxigenic isolates to inhibit toxin production by aflatoxigenic strains on infected maize kernels. It was shown that the atoxigenic strain (KSMO12) could inhibit the aflatoxigenic strain (KSM014) depending on the atoxigenic concentration during infection. To our knowledge, this is the first reported study for A. flavus genetic diversity, variation and distribution in Nandi, Homa Bay and Kisumu regions in comparison to and could assist researchers in the selection of biocontrol strategies to mitigate aflatoxin contamination, especially in Makueni and neighbouring regions.

No description available.

Experiments were conducted to study the ontogeny, kinetics, and the influence of aflatoxin B1 on antibody response to sheep erythrocyte (SRBC) antigen in White Leghorn chickens. In the first experiment, chickens from the parental lines and reciprocal crosses between them were fed diets containing graded levels, from 0 to 5697 ppb of aflatoxin B₁. Aflatoxin depressed body weights, feed consumption and feed conversion, with feed conversion being depressed less than either body weight or feed consumption. Although there were no differences among aflatoxin levels for body core temperatures, levels of 1830 ppb and higher caused progressive decreases in surface temperatures. Heterophilia, lymphopenia and reduced liver metabolism were observed at the 5697 ppb level. Although bursa and thymus weights were smaller in the aflatoxin-fed birds, there was no reduction in their SRBC antibody levels. The second experiment was designed to measure primary and secondary antibody response to intravenous immunization of SRBC antigen. Treatments included immunization at the dosage of SRBC antigen under which selection was practiced, and higher and lower concentrations. Although the dosage of primary immunization influenced the magnitude of the secondary response within population-primary dosage correlations between peak primary and secondary antibody response were not different from zero. Differences among populations in antibody levels appeared as early as day 4 and persisted until day 24 post-primary immunization. Yet, the general response patterns were the same for all populations with respective peaks occurring at the same time. The ontogeny of post-hatching production of antibody SRBC antigen and growth of bursa, thymus and spleen were measured in the third experiment. Both parental lines and reciprocal crosses between them reached serological maturity by 14 days of age. By 7 days, there were differences among populations for frequency of responders to SRBC antigen and magnitude of titers, inferring genetic variation for both the event and subsequent levels of antibody production. / Ph. D.

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Aflatoxins and fumonisins are among the most toxic mycotoxins. They are a significant risk factor for a cocktail of chronic health conditions including cancer of the liver, oesophagus and kidney, teratogenicity, neural tube defects, interference with lipid metabolism, a weakened immune system and a negative impact on micronutrient absorption in both man and animals. This study compared natural contamination of peanuts, peanut butter and sorghum from Gaborone, Botswana and Bulawayo, Zimbabwe with aflatoxins and fumonisins. In total 34 peanut samples, 34 sorghum samples and 11 peanut butter samples were collected randomly from retail shops and informal markets in the two cities. Fungal contamination was determined using standard mycology methods. Aflatoxin and fumonisin contamination was determined using HPLC-FLD. A. flavus/parasiticus species were detected in 66% and 100% of randomly analysed peanut samples from Bulawayo and Gaborone respectively and 27% (3/11) of peanut butter samples from Bulawayo. 67% of randomly analysed sorghum samples from Bulawayo showed A. flavus/parasiticus and Fusarium species contamination while none of the randomly analysed sorghum samples from Gaborone showed any fungal contamination. Furthermore aflatoxins were not detected in any of the sorghum samples; however 61% (11/18) of the Bulawayo sorghum samples showed fumonisin contamination (Range: 8 – 187 ng/g). Three of the peanut samples from Bulawayo were contaminated with aflatoxins (range: 6.6 – 622 ng/g) and no aflatoxins were detected in Gaborone peanuts. All 11 peanut butter samples from Bulawayo were contaminated with aflatoxins (Mean: 73.5 ng/g, Range: 6.8-250 ng/g) and AFB1 was the most prevalent. These preliminary results indicate that peanut butter and peanuts from Bulawayo are contaminated with high levels of aflatoxins. Stricter policing of regulations should be implemented to ensure compliance by manufacturers and public health interventions implemented in vulnerable communities. / Life & Consumer Sciences / M. Sc. (Life Sciences)

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Aflatoxins and fumonisins are among the most toxic mycotoxins. They are a significant risk factor for a cocktail of chronic health conditions including cancer of the liver, oesophagus and kidney, teratogenicity, neural tube defects, interference with lipid metabolism, a weakened immune system and a negative impact on micronutrient absorption in both man and animals. This study compared natural contamination of peanuts, peanut butter and sorghum from Gaborone, Botswana and Bulawayo, Zimbabwe with aflatoxins and fumonisins. In total 34 peanut samples, 34 sorghum samples and 11 peanut butter samples were collected randomly from retail shops and informal markets in the two cities. Fungal contamination was determined using standard mycology methods. Aflatoxin and fumonisin contamination was determined using HPLC-FLD. A. flavus/parasiticus species were detected in 66% and 100% of randomly analysed peanut samples from Bulawayo and Gaborone respectively and 27% (3/11) of peanut butter samples from Bulawayo. 67% of randomly analysed sorghum samples from Bulawayo showed A. flavus/parasiticus and Fusarium species contamination while none of the randomly analysed sorghum samples from Gaborone showed any fungal contamination. Furthermore aflatoxins were not detected in any of the sorghum samples; however 61% (11/18) of the Bulawayo sorghum samples showed fumonisin contamination (Range: 8 – 187 ng/g). Three of the peanut samples from Bulawayo were contaminated with aflatoxins (range: 6.6 – 622 ng/g) and no aflatoxins were detected in Gaborone peanuts. All 11 peanut butter samples from Bulawayo were contaminated with aflatoxins (Mean: 73.5 ng/g, Range: 6.8-250 ng/g) and AFB1 was the most prevalent. These preliminary results indicate that peanut butter and peanuts from Bulawayo are contaminated with high levels of aflatoxins. Stricter policing of regulations should be implemented to ensure compliance by manufacturers and public health interventions implemented in vulnerable communities. / Life and Consumer Sciences / M. Sc. (Life Sciences)

Thesis (D.Tech.: Biotechnology)-Dept. of Biotechnology, Durban Institute of Technology, 2005 xx, 175, [14] leaves : ill. ; 30 cm / Previous studies indicate that a mutation in the non-oncogenic p53 gene is epidemiologically linked to human HCC (Ozturk, 1991; Chan et al., 2003). Hsu et al. (1991) found this link in Chinese, South African and Asian patients and Hollstein et al. (1993) found the same gene mutation in Taiwanese patients. The incidence of these aberrations is reported to be about 20- 50% in HCC’s (Kishimoto et al., 1997). There is sufficient evidence to indicate that carotenoids in addition to their well known antioxidant properties (Paiva and Russel, 1999), also affect intercellular communication, immune responses, neoplastic transformations and growth control, and cellular levels of enzymes that detoxify carcinogens (Zhang et al., 1991; Brockman et al., 1992; Pryor et al., 2000). To date studies carried out have used the rat (Foote et al., 1970; Gradelet et al., 1998) and the mule duckling model (Cheng et al., 2001) to show the protective effect of these carotenoids against AFB1 exposure. Of the well known carotenoids, lycopene and beta- carotene occur in abundance in fruits and vegetables and are safe for human consumption. Aflatoxin B1 frequently induces mutations of the p53 gene which is linked to HCC. Although there is much evidence from epidemiological studies linking the beneficial aspects of carotenoids to the prevention of cancer, the cellular and molecular mechanisms need to be understood in order to implement large scale intervention strategies to prevent AFB1 induced carcinoma. The use of chemical or dietary interventions to alter the susceptibility of humans to the actions of carcinogens and to block, retard or reverse carcinogenesis is an emerging chemoprotective strategy for disease prevention (Abdulla and Gruber, 2000; Kensler et al., 2003; Bingham and Riboli, 2004). Chemoprotection by natural products involves maintaining cellular integrity, preventing DNA alterations, activation of p53 suppressor protein and apoptosis. The aim of this study was thus to investigate the cellular and molecular mechanisms by which beta-carotene and lycopene may prevent the AFB1-induced toxic changes in human hepatocytes. In order to achieve this aim, the following objectives were set out: i. To optimise an in vitro system for the evaluation of AFB1 damage to cultured hepatocytes. ii. To determine the biochemical protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by measuring the mitochondrial activity, cell viability and ROS levels using appropriate enzyme assays and flow cytometry. iii. To determine the cellular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by studying the morphological changes at the structural and ultrastructural levels using phase contrast light and electron microscopy respectively. iv. To determine the molecular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by detecting apoptotic bodies as genomic markers and measuring the levels of p53 protein and AFB1-N7-guanine adducts produced.