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The Prevalence and Health Effects of Fungi and Mycotoxins in Food Commodities from Cameroon By NJOBEH Patrick BERKA A dissertation Submitted to the Faculty of Health Science, University of Johannesburg, South Africa, in fulfillment of the Requirements of an Award of a Doctorate Degree in Technology: Biomedical Technology SUPERVISOR Prof M. F. Dutton CO-SUPERVISOR Prof A. A. Chuturgoon July 2009 SUMMARY To determine the quality of human food commodities commonly consumed in Cameroon, various districts in the western highland (Bamenda and Kumbo) and tropical rain forest (Douala and Yaounde) regions were sampled. Two mycological investigations were conducted to evaluate the incidences of mycotoxigenic fungi (95 samples) and mycotoxins (82 samples). Serial dilution of ground samples was employed to isolate fungi, subculture on various culture media and fungal species were identified morphologically followed by molecular phylogenetic approach. In general, data obtained indicate samples from various geographical regions showed no consistent variation with regard to the type of fungal species. The mycobiota of food materials were characterized by a diversity of fungal species with the predominance of Aspergillus (125 isolates) followed by Penicillium (94 isolates) and Fusarium (52 isolates). The less predominant genera include Rhizopus (14 isolates) and the Alternaria (9 isolates). Aspergillus flavus and A. parasiticus occurred in 53 and 44% of the samples, respectively, with higher frequencies in maize than peanuts or beans and absent in rice, pumpkin seed and cassava products. Aspergillus fumigatus was detected in 20% of samples and A. niger in 18% of the samples. Aspergillus isolated less frequently included A. carbonarius A. awamori, A. oryzae and A. tamarii, A. pseudotamarii, A. ochraceus, A. ostianus, A. avenaceum, A. oryzae and A. variabile. Consistent results were observed for A. tamarii, A. pseudotamarii, A. ochraceus and A. ostianus with respect to substrate specificity. While A. tamarii, A. pseudotamarii and A. ochraceus were isolated only from peanuts, A. ostianus strains occurred only in bean samples. Penicillium contamination was dominated by P. polonicum and P. crustosum with incidence rates of 43 and 41%, respectively, with highest contamination levels registered in samples from Yaounde. Penicillium citrinum, P. purpurogenum, P. islandicum, P. aurantiogriseum, P. expansum were also inconsistently isolated from food samples. There was a relatively low incidence of Penicillium spp. in pumpkin seed and fermented cassava product samples. Of the 52 isolates of Fusarium recovered 19 were identified as F verticillioides, 9 as F. sp Cameroon II, 8 as F. oxysporum, 6 as F. sp Cameroon I, 3 as F. sp 25622, 2 as F. semitectum, 2 as F. sp Cameroon III, 1 as F. inflexum and another as F. graminearum. Fusarium verticillioides was recovered from maize but also occurred in bean, peanut and rice samples. ii Data obtained in this study also identified some cryptic species viz; F. sp Cameroon I, F. sp Cameroon II and F. sp Cameroon III. Fusarium was not isolated from pumpkin seed or fermented cassava product samples. Of particular concern was the increased prevalence of co- contamination of more than one species of fungi in similar samples, with only 4 of the surveyed samples had no fungi. The isolates from food materials were investigated for laboratory production of their respective mycotoxins. A number of mycotoxins of toxicological importance, including several unknown metabolites, were identified. In terms of their potentials to produce mycotoxic compounds, Aspergillus, Penicillium and the Fusarium represented the most important genera, with most of the isolates producing their respective mycotoxins. Strains of A. flavus and A. parasiticus tested were aflatoxin producers, with A. flavus producing aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2), while A. parasiticus produced AFB1, AFB2, aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). Aspergillus ochraceus was the only ochratoxin A (OTA) producer found in this study. Isolates of P. crustosum and P. polonicum found produced an uncharacterised metabolite giving a blue-green fluorescent spot under UV-radiation. The occurrence of this metabolite in food samples was common and was therefore further studied. Among the Fusarium species, F. verticillioides was a producer of fumonisin B1 (FB1), zearalenone (ZEA), deoxynivalenol (DON) and T-2 toxin in addition to the new Fusarium species viz; F. sp. Cameroon II and F. sp. Cameroon I. Mycotoxicological survey of similar samples was also performed and data obtained from 82 samples. Forty-four samples (54%) contained FB1, 63 samples (77%) contained ZEA, 62 samples (76%) contained DON, 63 samples (77%) contained AF and 3 samples (4%) contained OTA. Fumonisin B1 in maize averaged 3,415 ppb in 31 of the 40 maize samples analyzed when compared to peanut, bean, soybean and rice with average FB1 contents of 1,498, 1,351, 365 and 20 ppb, respectively. Zearalenone mean levels of 87 ppb in peanuts and 57 ppb in maize were much higher than the 43, 34 and 3 ppb for beans, rice and soybean, respectively. Although concentrations were low in this study, frequency of DON in similar samples was high with mean content of 77 ppb recorded in peanut samples. Total aflatoxin (AF) contamination was highest in peanuts with a mean content of 6 ppb followed by beans (2 ppb). Frequencies of AFB1 and AFG1 in samples were low but exceeded acceptance levels (5 ppb) in 10 and 2 iii samples, respectively. Mean level of OTA was 5 ppb. Although the incidence of fungal contamination was significantly less than that of mycotoxins in similar samples, it was noted that the incidence of fungi was partially associated with those of their corresponding mycotoxins. The uncharacterised metabolite produced by P. polonicum (m/z 390.2770 and empirical formula C24H38O4) was found in 37 samples (45%) including 17 maize, 10 peanut, 7 beans, 2 soybeans and 1 rice. Co-contamination of samples with two or more mycotoxins in the same sample was also noted with only 5 samples having only one mycotoxin present. The cytotoxic potentials of OTA, T-2 Toxin and the uncharacterised metabolite (Mycotoxin X) of P. polonicum were compared ―in vitro‖ on human lymphocyte cells. Cell viability was strongly influenced by both the type and concentration of toxin exposure when compared to the control. A significant mycotoxin-induced-cell mortality provoked by dose increment was observed. Accordingly, the cell viability decreased (P<0.001) significantly with increasing concentration levels of all the mycotoxins tested. Increasing the concentration of T-2 toxin from 0.15 to 5.0 µg/ml significantly (p<0.001, R2 = 0.79) decreased cell viability from 64.8 to 23.6%. Increasing the level of the Mycotoxin X, from 0.15 to 50 µg/ml resulted in a significant (p<0.001, R2=0.79) decrease in cell viability from 63.3 to 25.8% and a similar trend of 55 to 12.4% (p<0.01, R2 = 0.80) was observed for OTA. The lowest cell viability was recorded when cells were exposed to 5.0 µg/ml of OTA, while cells were most viable when exposed to 1.56 µg/ml of T-2 toxin. To test the susceptibility of the fungal isolates to fungicidal control, a disc application technique was employed ―in vitro‖. Against fungicide application, F. verticillioides was the most susceptible, while F. oxysporum was found to be the most resistant among all species. Among the fungicides tested, tebuconazole against F. verticillioides was the only fungicide that effectively inhibited fungal growth by more than 50% when administered at recommended level by Day 4. Carbendazim/flusilazole, thiabendazole, tebuconazole and mancozeb were the most effective from a broad spectrum viewpoint, providing significant differences (p<0.001) on the %MIZ of fungal mycelia with long lasting potentials exhibited by carbendazim/flusilazole, thiabendazole and tebuconazole. Conversely, the protective effects of azoxystrobin, fludioxonil, iprodione, carboxin and carboxin/thiram against fungal growth were noted in this study. iv The results obtained in these studies established an association between fungal contamination, mycotoxin production in human foods from Cameroon, and possible human health implications. Data from these studies have highlighted some comparative information on the current situation of food quality, while screening at least two new species of Fusarium and identifying a novel Penicillium mycotoxin of toxicological significance. Although the levels of mycotoxins were low in this study, their presence indicates the need for further monitoring of the food supply, development of regulatory levels for mycotoxins, and the need for research to develop control protocols. v TABLE OF CONTENT SUMMARY…………………………………………………………………..ii TABLE OF CONTENT………………………………………………….......vi LIST OF TABLES…………………………………………………………....xi LIST OF FIGURES………………………………………………………….xii