Evaluate of amplificability of genomic DNA from two in-house protocols of conserved sequence by biosynthesis pathway of filamentous fungi producers of aflotoxins

In the present study, the polymerase chain reaction (PCR) was evaluated as a molecular technique to identify the aflotoxins filamentous fungi producers. The PCR was carry out using a specific pair of primers for b-tubulin amplification, that has a high conserved sequence in filamentous fungi. It was tested two DNA extraction methods: Lee et al. and Weising et. al. both methods were efficient in DNA isolation. The results showed that using Lee et. al. method was possible to extract DNA with better quality, and the PCR runs presented the expected band for b-tubulin. These results showed that the lee et al. method and PCR is adequate for Aspergillus sp. detection. There is a perspective to improve the detection using specific primers to amplify genes related to aflotoxin biosynthesis, allowing the development of a molecular method to evaluate the risk contamination of this substance in food

Fungos micotoxigênicos e ocratoxina a em cafés com permanência prolongada na planta e no solo, colhidos nas regiões do cerrado mineiro e baiano

In Brazil, the regions called ‘Cerrado’ in Minas Gerais and Bahia states are expressive coffee producers and stand out for productivity and quality of grain produced. The bloom unevenness of coffee in Brazil leads to fruit with various stages of maturity at the harvest period. As a result, some producers use low quality grains with long soil or plant contact and mix them with those of quality to compose the harvest. The objective of this work was to study coffee fruits with soil contact of up to 90 days evaluating ochratoxin A (OTA) levels, humidity dynamics and water activity in coffee fruits harvested every 30 days. The coffee with prolonged stay on the plant, showed no major variations in humidity and water activity levels during the 120 days studied. However, in coffee with prolonged stay on the soil, after 90 days, the humidity and water activity levels varied dramatically according each region. During this period, the humidity and water activity were 14.15 and 0.74%, 6.64% and 0.63 for the Cerrado Mineiro and Baiano, respectively. Despite the coffee with prolonged stay in the plant having been heavily colonized by Aspergillus ochraceus G. Wilh. (1877),  OTA contamination was not detected, but in coffees with prolonged stay on the soil, high levels of OTA were observed, 49.42 and 30.93 μg.kg -1, for the Cerrado Mineiro and Baiano, respectively. There was OTA  production even in the grains with low water activity, indicating how the complexity of the system in the field interferes with the micotoxigenic dynamic fungi growth and consequently in OTA production.No Brasil, as regiões do Cerrado Mineiro e Baiano são destaque pela produtividade e qualidade dos grãos produzidos. A florada desuniforme do café no Brasil implica em frutos com diversos estágios de amadurecimento no período da colheita. Assim, frutos não colhidos oportunamente permanecem na planta ou caem no solo, e quando aproveitados farão parte de uma safra de baixa qualidade. Portanto, foi objetivo deste trabalho estudar frutos de café com exposição prolongada na planta e no solo, avaliando a colonização por fungos micotoxigênicos, a produção de ocratoxina A (OTA) e a dinâmica de umidade e atividade de água nos frutos nessas regiões produtoras. O café com permanência prolongada na planta não apresentou grandes variações nos teores de umidade e atividade de água durante os 120 dias estudados. Entretanto, o café com permanência prolongada no solo, apresentou, após 90 dias, variação drástica nos teores de umidade e atividade de água entre as regiões estudadas. Nesse período, a umidade e atividade de água foram de 14,15% e 0,74, 6,64% e 0,63 para o Cerrado Mineiro e Baiano, respectivamente. Apesar do café com permanência prolongada na planta ter sido intensamente colonizado por Aspergillus ochraceus G. Wilh. (1877), não foi detectada a presença de OTA. No café com permanência prolongada no solo detectaram-se níveis muito elevados, 49,42 e 30,93 µg.kg-1 de OTA, no Cerrado Mineiro e Baiano, respectivamente. Pode-se constatar que independente da região de interesse, cafés com permanência prolongada na planta ou no solo interferem decisivamente na qualidade do café colhido

Predominant mycobiota and aflatoxin content in Brazil nuts

Abstract The Brazil nut (Bertholletia excelsa) is an economically important product to the Brazilian Amazon. Currently, its marketing is being affected by the high incidence of aflatoxins (AF) produced by potentially aflatoxigenic fungi associated with its seeds. In this context, the purpose of this study was to determine which part of the nut contributes to contamination by aflatoxins and to identify the mycobiota in Brazil nut samples. Unshelled and shelled nuts were analyzed by measuring the total count of filamentous fungi (Aspergillus sections Flavi, Nigri and Circumdati) in sanitised and non-sanitised treatments. The isolates identified as Aspergillus section Flavi, the major producers of AF, were plated for determination of their aflatoxigenic potential. To perform the AF analysis, samples of Brazil nuts were treated separately. The AF from the shell and kernel were extracted by chloroform and analysed by the HPLC-FD system in isocratic mode. The Aspergillus section Flavi count was 21.67% lower. The production of AF by the isolated fungi was 30% for sanitised and 23.8% for non-sanitised samples. The concentrations obtained of AFB1 and AFG1 were higher than those of AFB2 and AFG2. The AFB1 concentrations of shelled nuts and shell samples were 35.0 and 1.78 lg/kg, respectively. AFB2 and AFG2 were detected only in shelled nut samples. The HPLC-FD analysis presented limits of detection (LOD) and quantification (LOQ) of 0.2 and 0.4 lg/kg, respectively