Black Aspergillus species as ochratoxin A producers in Portuguese wine grapes

To evaluate the incidence of fungi producing ochratoxin A (OA) in Portuguese wine grapes, a survey was conducted in 11 vineyards, from four winemaking regions each with distinct climatic conditions. From setting to the harvesting period, a total of 1650 berries were sampled by plating methods. Out of 370 aspergilli and 301 Penicillium strains isolated, 14% of the aspergilli were OA-producing strains. None of the penicillia were OA-producing strains. The black aspergilli were predominant (90%). All Aspergillus strains were tested in vitro for OA production and all were preserved in the Micoteca da Universidade do Minho (MUM) culture collection. Most of the Aspergillus carbonarius (97%) and 4% of the Aspergillus niger aggregate strains were OA producers. Almost all ochratoxigenic strains were isolated at harvest time, mainly in the regions with a Mediterranean climate. In the vineyards sampled, the percentage of colonized berries with ochratoxigenic strains was up to 38%. The vineyards from the region with Atlantic influences, with high rainfall, exhibited the lowest occurrence of Aspergillus and ochratoxigenic strains, 0% to 10% and 0% to 2% colonized berries, respectively. Data obtained here supports the hypothesis that A. carbonarius and occasionally A. niger, are the main producers of OA in grapes. In this study, the highest incidence of these fungi occurred in vineyards with a Mediterranean climate.European Commission (EC) - Quality of Life Programme (QoL) Fundacão para a Ciência e Tecnologia - SFRH/BD/1436/200

Identification of aflatoxigenic and non‐aflatoxigenic strains of Aspergillus section Flavi isolated from Portuguese almonds

Aspergillus subgenus Circumdati section Flavi, also referred to as the A. flavus group, has attracted worldwide attention for its industrial use and toxigenic potential. Section Flavi is divided in two groups of species. One includes the aflatoxigenic species A. flavus, A. parasiticus and A. nomius, which cause serious problems in agricultural commodities, and the other one includes the non‐aflatoxigenic species A. oryzae, A. sojae and A. tamarii, traditionally used for production of fermented foods. Differentiating aflatoxigenic from non‐aflatoxigenic species and strains in food commodities is of major importance in food quality control. A polyphasic approach consisting of morphological, chemical and molecular characterization was applied to 31 isolates of Aspergillus Section Flavi originating from Portuguese almonds, with the aim of characterizing and identifying aflatoxigenic and non‐aflatoxigenic strains. On the basis of morphological characters, we found two distinct groups among the population under study: 58% were classified as A. parasiticus and the remaining 42% were classified as A. flavus. Chemical characterization involved the screening of the isolates for aflatoxins B (AFB) and G (AFG), and also for cyclopiazonic acid (CPA), by HPLC. All A. parasiticus isolates were strong AFB and AFG producers, but no CPA production was detected. The A. flavus isolates showed to be more diversified, with 77% being atoxigenic, whereas 15% produced CPA and low levels of AFB and 8% produced the 3 groups of mycotoxins. Molecularly, two genes of the aflatoxin biosynthetic pathway, aflD (=nor1) and aflQ (=ord1= ordA) were tested for presence and expression (by PCR and RT‐PCR, respectively). The presence of both genes did not correlate with aflatoxigenicity. aflD expression was not considered a good marker for differentiating aflatoxigenic from non‐aflatoxigenic isolates, but aflQ showed a good correlation between expression and aflatoxin‐production ability.Fundação para a Ciência e a Tecnologia (FCT) - bolsa SFRH/BD/28332/200

Fourier transform infrared as a powerful technique for the identification and characterization of filamentous fungi and yeasts

Fourier transform infrared is considered a powerful technique for characterizing chemical compositions of complex probes such as microorganisms. It has successfully been applied to fungal identification. In this paper, the current state of identification and characterization of filamentous fungi and yeasts by Fourier transform infrared is reviewed.European Community - Seventh Framework Program (FP7,2007-2013)Research Infrastructures Action -grant agreement No. FP7-228310 (EMbaRC project

White-rot fungi capable of decolourising textile dyes under alkaline conditions

Twelve white-rot fungal strains belonging to seven different species were screened on plates under alkaline condition to study the decolourisation of the textile dyes Reactive Black 5 and Poly R-478. Three strains of Trametes versicolor (Micoteca da Universidade do Minho (MUM) 94.04, 04.100 and 04.101) and one strain of Phanerochaete chrysosporium (MUM 94.15) showed better decolourisation results. These four strains were used for decolourisation studies in liquid culture medium. All four selected strains presented more efficient decolourisation rates on Reactive Black 5 than on Poly R-478. For both dyes on solid and liquid culture media, the decolourisation capability exhibited by these strains depended on dye concentration and pH values of the media. Finally, the decolourisation of Reactive Black 5 by T. versicolor strains MUM 94.04 and 04.100 reached 100 %. In addition, the highest white-rot fungi ligninolytic enzyme activities were found for these two strains

Polyphasic approach including MALDI-TOF mass spectrometry to characterise aflatoxigenic species of Aspergillus section flavi

Aflatoxins are toxic compounds which are produced as secondary metabolites by the fungi Aspergillus flavus, A. parasiticus and A. nomius growing on a variety of food products and are known to be carcinogenic, mutagenic, teratogenic and immunosuppressive1,2. Aspergillus is a large genus, with a complex taxonomy. The genus is easily identified by its characteristic conidiophore, but species identification and differentiation is complex, mainly because it is traditionally based on a range of morphological features. One includes the aflatoxigenic species referred above A. flavus, A. parasiticus and A. nomius, which cause serious problems in agricultural commodities, and the other one includes the non-aflatoxigenic species A. oryzae, A. sojae and A. tamarii, traditionally used for production of fermented foods. Species from A. flavus group are morphologically and genetically very similar, and are therefore difficult to differentiate by both cultural and molecular methods. Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry has already shown high potentialities in discriminating very closely related taxa. In this work is presented a polyphasic approach including MALDI-TOF MS to discriminate A. flavus group strains.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/28332/200

Identification and characterization of Aspergillus flavus and aflatoxins

Aspergillus flavus is the main producer of the well known carcinogenic aflatoxins. The presence of this fungus and aflatoxins is of huge concern in terms of food safety. The identification of A. flavus is not straightforward due to similarities with closely related species (e.g. A. parasiticus and A. nomius). Also, from the biochemical point of view the closely-related species are able to produce different mycotoxins. In order to clarify the differentiation between species the identification schemes is revisited. Selective media, data from mycotoxins production and molecular biology tools are discussed in order to clarify the concept of A. flavus species.Fundação para a Ciência e a Tecnologia (FCT

Fungi in bottled water: a case study of a production plant

A one year fungal survey of a water bottling plant was conducted in order to evaluate the incidence and fluctuations of the biota. The dominant fungal genera in order of highest numbers were Penicillium, Cladosporium and Trichoderma followed by Aspergillus, Paecilomyces, and others. As expected, highest number of isolates collected were during the summer months, particularly May and June. Indeed during these two months there were more fungi present in the water after it had passed through the filtration system (0.4μm filter), indicating that during those times of the year when fungal contamination is high, filters should be changed on a more regular basis. In order to assess whether contamination was single or multi-loci, molecular methods based on PCR were used. Overall fungal contamination arose from multiple sources. Some fungal strains were very “alike” and were detected during different sampling times, indicating that some strains were endemic to the plant. There was little evidence to suggest that fungi detected in the source water passed through to other parts of the plant. However, there was evidence that fungal strains isolated from the water filter were detected elsewhere in the factory, confirming the need to change filters more regularly during periods of high fungal contamination. In order to improve quality control a HACCP programme was implemented and Best Practice Guidelines introduced.Control of Mycological Contaminations in Bottled Water (COMBOW) - CRAFT/QLK1-2002-70843 contract

Contributions of micoteca da Universidade do Minho to food mycology

Micoteca da Universidade do Minho (MUM) is a collection of fungi established in 1996 where research into secondary metabolites in the form of mycotoxins is paramount. An objective is to maintain and provide authentic strains for research in biotechnology, life sciences and challenge testing. Knowledge, information and training in mycology are promulgated by the biological resource centre. MUM is involved in projects which address the health and quality risks from fungal contamination of grape products (e.g. wine), apples, cheese, chillies, nuts and corn: Fungi in drinking water are of particular concern. Aspergillus ochraceous, Aspergillus flavus, Penicillium expansum and Fusarium graminearum especially are relevant to our work: Associated mycotoxins are ochratoxin A, aflatoxins, patulin, citrinin and zearalenone. We study the earthy off-flavours in wine from Botrytis cinearum and P. expansum. The analytical techniques employed are PCR, HPLC, GC-MS and MALDITOF MS for strain characterization. MUM participates in several European and national projects. Post-graduate studies on mycotoxigenic fungi and food security particularly are important. A key objective is international collaboration. MUM strains are well characterized for toxigenicity, making the collection relevant to its mission and industry. This presentation will describe the progress made in this innovative Portuguese laboratory over 10 years

Food mycology : from the fork to the farm

Filamentous fungi are ubiquitous in nature and are responsible for producing mycotoxins in agricultural crops. Fungi and mycotoxins are considered generally as being natural and unavoidable [1]. The control and detection of mycotoxins is a continuous process in commodity production. Since mycotoxins can become established and remain within the commodity anywhere throughout the production, storage, transportation and processing chain, there is a need to study all the food chain: from the fork to the farm. The control over the presence of mycotoxins involves several competencies, such as taxonomy skills, ability to evaluate mycotoxins production by fungi, and ability to quantify mycotoxins in commodities. The prevention of mycotoxin formation is achieved by influencing environmental conditions through management of agricultural practices prior to harvest. After harvest, two overriding factors for storage are water activity (aw) and temperature. Control over these parameters may lead to the prevention of fungi growth and of mycotoxins accumulation. Strategies followed to have control over the presence of mycotoxins in commodities will be illustrated by presenting case studies: (i) ochratoxin A in wine; and (ii) aflatoxins in maize and in nuts