Browsing by Author "Pedro Vicente Martinez-Culebras"

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    Evaluation of DNA extraction methods for PCR detection of fungal and bacterial contamination in cocoa extracts
    (2009) Marta Tortajada; Pedro Vicente Martinez-Culebras; Veronica Navarro; Honorato Monzo Daniel Ramon
    Direct and sensitive PCR detection of contaminant microXora in cocoa extracts is aVected by the quality of the template DNA. This study compares the eYcacy of Wve diVerent commercial DNA extraction methods, selective enrichment broths and use of glycolitic enzymes to obtain quality DNA for PCR detection of both fungi and bacteria in artiWcially inoculated cocoa extract samples. PCR-based methods were applied to detect contaminant microXora in cocoa extracts using as model organisms: Aspergillus nidulans, Bacillus subtilis, Escherichia coli and Salmonella enterica. The quality of the extracted DNA was assessed in terms of PCR inhibitor content with results indicating that the HighPure PCR template (Roche) kit was the best methodology under the conditions assayed. PCR protocols using this commercial kit and a combination of glycolitic enzymes and enrichment procedures gave a detection limit of 100 conidia/g and 100 cfu/g for Wlamentous fungi and bacteria, respectively. The selected extraction and PCR procedures were also tested to assess their suitability for detecting Wlamentous fungi and bacteria on an industrial scale. They were sensitive enough to detect fungal and bacterial contaminants within the legally required limits. The results obtained with the molecular approach were in agreement with those of standard microbiological tests but require a considerably shorter analysis time. Thus, the molecular approach provides a sensitive and rapid alternative to check for microbial contamination in cocoa extracts.
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    ITS-RFLP characterization of black Aspergillus isolates responsible for ochratoxin A contamination in cocoa beans
    (2009) Fatima Bisbal; Jose Vicente Gil; Daniel Ramon; Pedro Vicente Martinez-Culebras
    In the present study, ochratoxigenic mycobiota in cocoa beans was identiWed at species level by digestion of the ITS products using the endonucleases HhaI, NlaIII and RsaI. Of the 132 isolates of Aspergillus section Nigri collected from cocoa beans, 89 were identiWed as A. tubingensis, 27 as A. niger, 10 as A. tubingensis-like and 6 as A. carbonarius. No variation was observed between RFLP patterns (C, N, T1 and T2) described previously for grape isolates and those of the cocoa isolates analysed. With respect to OTA-producing fungi, a high percentage of black aspergilli (50.7%) was able to produce OTA. Additionally, most of the OTA-producing isolates were of moderate toxigenicity, producing amounts of OTA from 10 g g¡1 to 100 g g¡1. Percentages of OTA-producing isolates in the A. niger aggregate were higher than in other substrates, ranging from 30% to 51.7%. Furthermore, the detected levels of OTA production in the A. niger aggregate, particularly in A. tubingensis species was higher than in A. carbonarius, ranging from 0.7 g g¡1 to 120 g g¡1 (mean 24.55 g g¡1). Due to the high occurrence, percentage of ocratoxigenic isolates and their ability to produce OTA, isolates belonging to the A. niger aggregate could be considered as the main cause of OTA contamination in cocoa beans used for manufacturing cocoa products.