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 1,650 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.

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that have been detected in food commodities, including grapes and wine. A survey was conducted to assess mycotoxin-producing fungi in grapes destined for wine production. The mycotoxigenic capacity of the isolates was tested in culture media. Grapes were analyzed by plating methods from 4 Portuguese wine-growing regions at 3 maturation stages (pea berry, early veraison and ripe berry) between 2001 and 2003. From the 10,602 strains detected and identified, the most frequent genera were Cladosporium (25%), Alternaria (24%), Botrytis (15%), Penicillium (9%) and Aspergillus (8%). Most (92.0%) were non-mycotoxigenic or produced mycotoxins of unknown relevance to health. Potential producers of ochratoxin A (OTA) (Aspergillus niger aggregate, 5.4%, Aspergillus carbonarius, 0.6%) and trichothecenes (Fusarium spp., 0.4%; Trichothecium roseum, 0.8%) were the most frequent mycotoxigenic species isolated from grapes. OTA was detected in all cultures of A. carbonarius and 4% of A. niger aggregate strains. There was potential for OTA and trichothecene production in grapes by A. carbonarius and T. roseum, respectively, prior to harvest time. Data presented herein indicate that A. carbonarius is most likely to occur in vineyards with Mediterranean climates, while T. roseum is more likely to occur in more temperate climates, and is associated with gray rot. The present work emphasizes the need to use grapes under good condition so as to reduce the risk of contamination with mycotoxigenic fungi and subsequent mycotoxin occurrence in wine.

Ochratoxin A is a mycotoxin present in several food products for which levels should be reduced. Chemical, physical, and biological methods have been proposed for the detoxification of mycotoxins, biological methods being the more promising ones. In this report, filamentous fungi isolated from Portuguese grapes were assessed for ochratoxin A degradation capabilities. It was observed that 51 of the 76 tested strains, predominantly aspergillus species, were able to degrade more than 80% of ochratoxin A added to the culture medium and that the most potent species (more than 95% of initial amount) were the black aspergilli, A. clavatus, A. ochraceus, A. versicolor, and A. wentii. Other fungi frequently isolated from grapes, such as Alternaria, Botrytis, Cladosporium, and Penicillium, also showed significant degradation capabilities. It was observed that the compounds obtained from the degradation of ochratoxin A by black aspergilli and by A. ochraceus and A. wentii strains were different.

A three-year study was undertaken to investigate the fungal species present on the surface of grape berries from Portuguese vineyards in four winemaking regions. Emphasis was given to Aspergillus and Penicillium species due to their relevance for mycotoxin production. From the 3517 fungal strains detected 27 genera were identified. The region of origin markedly influenced the spoilage fungal population to which berries are exposed. The main differences found were in the incidence of A. niger aggregate, Botryis cinerea and Penicillium species (P. brevicompactum, P. citrinum, P. glabrum/spinulosum, P. expansum, P. implicatum and P. thomii). In more humid climates, Botrytis seems to be the main pathogen and spoiling agent, and the incidence of black Aspergillus is minimal. The most important mycotoxin-producing species found was A. carbonarius, which is an ochratoxin A producer. The present study provides a detailed description of the fungi found on the berry surface of Portuguese grapes and shows the Aspergillus and Penicillium species, which vary significantly by geographic origin. This is of crucial importance to understand fungal hazards for grapes and wine and to the knowledge of field ecology of the species.

Despite several studies reporting Penicillium as one of the most frequent fungal genera in cork planks, the isolates were rarely identified to species level. We conducted a detailed study to identify Penicillium species from the field to the factory environment prior to and after boiling the cork planks. A total of 84 samples were analyzed. Of the 486 Penicillium isolates phenotypically identified, 32 representative or unusual strains were selected for identification by multilocus DNA sequence type. Cork proved to be a rich source of Penicillium biodiversity. A total of 30 taxa were recognized from cork including rarely seen species and 6 phylogenetically unique groups. Spores of some species lodged deep in cork can survive the boiling process. P. glabrum, P. glandicola and P. toxicarium, species with high CFU numbers in the field, are still frequently present in cork after boiling. Other species are killed by the boiling treatment and replaced by Penicillium species originating from the factory environment. Species known to contribute to cork taint were isolated at all stages. Good manufacturing practices are necessary at all stages in the preparation of cork planks to minimize the load of Penicillium species that produce cork taint.

Cheese ripening rooms have an unusual environment, an environment that encourages mold growth. Ozone has been applied in various ways in the food industry. One useful advantage of ozone is that it inactivates molds. In this study, a cheese ripening room was ozonated, and the effectiveness of this treatment was evaluated both in air and on surfaces through sampling on a weekly basis over a 3-month period. The results obtained indicate that ozone treatment reduced the viable airborne mold load but did not affect viable mold on surfaces. Only by wiping the surfaces with a commercial sanitizer was it possible to decrease the viable mold load on surfaces. To improve overall hygiene in the ripening room, a combination of cleaning regimes is recommended. The mold genera occurring most frequently in the air of the cheese ripening room were Penicillium, Cladosporium, and Aspergillus, which accounted for 89.9% of the mold isolates. Penicillium and Aspergillus were identified to the species level, and data showed that P. brevicompactum and P. aurantiogriseum, as well as A. versicolor, were the species most frequently isolated.

As part of a study on the ochratoxin producing mycoflora of grapes, several Aspergillus strains were isolated and tested for their ochratoxin A (OTA) producing abilities. Aspergillus strains of the section Nigri, which did not produce detectable amounts of OTA but which had a similar morphology to A. carbonarius, were isolated from wine grapes and/or dried vine fruit in Portugal and Spain. These strains, however, have characters that allow morphological distinction from the other species in the section, particularly the conidia size (5-7 microm), which allows separation of the species from the two most common biseriate species in section Nigri: A. carbonarius (7-9 microm) and A. niger and its aggregate species (3-5 microm). The strains are described here as belonging to a new species, named A. ibericus. The validation of this new taxon is supported further by analysis of the ITS-5.8S rDNA and calmodulin gene sequences and by analysis of the amplified fragment length polymorphism (AFLP) patterns, which were consistent in separating these strains from other species in the section. A. ibericus strains do not produce OTA therefore they are interesting for biotechnological exploration because many metabolites with commercial value are produced by other species in the section.

A total of 93 Portuguese grape samples destined for wine production were examined for the presence of ochratoxin A (OTA) and the OTA producing fungi Aspergillus carbonarius and A. niger aggregate. Samples came from 11 vineyards from four winemaking regions in the North and South of the Portuguese mainland, during the harvest seasons of 2001, 2002 and 2003. Grapes were examined at 3 maturation stages, from setting to the harvesting period, to evaluate when contamination with OTA producing fungi and OTA synthesis occur. The detection of fungi in grape samples was made by plating methods with and without surface disinfection. OTA was formed by 14% of the 650 isolates tested. Most of the OTA producing strains (96%) were isolated at harvest time. At this stage, the percentage of grape samples with OTA producing strains detected without surface disinfection was 56%. With surface disinfection, A. carbonarius was isolated from 10% of the samples. OTA was detected in grapes at the 3 maturation stages. The average OTA concentrations in 60 samples at pea berry (28 samples), early veraison (22 samples) and ripe berry (20 samples) were 263, 149 and 35 ng/kg, respectively. Experiments with an A. carbonarius strain demonstrated that OTA production differs significantly with the composition of the berries at different maturation stages (P<0.001), with a mean value of OTA production at pea berry, early veraison and ripe berry of 3402, 1530 and 22 mug/kg, respectively. The production of OTA by A. carbonarius was correlated positively and negatively with the total acidity of grapes (r(s)=0.855, P<0.001) and reducing sugars content (r(s)=-0.835, P<0.001), respectively. Our data demonstrate that OTA synthesis in grapes occurs since early maturation stages.