European research on ochratoxin A in grapes and wine

European wine production represents about 70% of world production and thus is an important export commodity. Ochratoxin A (OTA) was first detected as a wine contaminant in 1996 and the role of Aspergillus section Nigri and A. carbonarius in OTA production discovered in Europe in 1999. Subsequently Europe-wide surveys have shown that A. carbonarius is predominantly responsible for OTA contamination of grapes, wine and vine fruits. Analyses of wine samples throughout Europe have shown that there is a gradient in OTA concentration with a decrease from red, to rose and to white wines. The latitude of production is an important factor in determining risk from OTA contamination. Some geographic regions in Southern Europe are more prone to contamination with the toxigenic species and OTA. Ochratoxin A has also been found in much higher concentrations (max. 53 mug/kg) in dried vine fruit than in wine suggesting that A. carbonarius can dominate the drying vine fruit ecosystem. There is a significant lack of knowledge in Europe on conducive climatic conditions preharvest and their relationship with levels of risk from OTA contamination in grapes and their fate in wine production. This needs to be integrated with cultivation system to maximise the prevention of OTA entering this food chain.     

Occurrence of ochratoxigenic fungi and ochratoxin A in grapes from a Tunisian vineyard

The occurrence of ochratoxin A (OTA) and the identification of the ochratoxigenic microbiota in Tunisian grapes were studied for the first time. Black aspergilli were the dominant genus among the filamentous fungi isolated from grapes and were the only potential OTA-producing fungi found. The most abundant species were member of Aspergillus niger aggregate (63%) and Aspergillus carbonarius (36%). Uniseriate aspergilli were rarely present (1%). Of the A. carbonarius isolates, 97% were OTA positive but only 3% of the A. niger aggregate isolates were OTA positive. During grape maturation, the frequency of black aspergilli increased due to increase of the numbers of A. carbonarius. Musts (n=24) obtained from grapes collected at the different sampling times were analyzed for their OTA content. Up to 37% of the musts contained OTA at levels varying between 0.59 and 2.57 microg/l. The amounts of OTA in musts increased as grapes matured. These results indicate that A. carbonarius is the main cause of OTA contamination of Tunisian grapes.     

Ochratoxin A-producing fungi from grapes intended for liqueur wine production

The ochratoxigenic mycobiota of grapes intended for liqueur wines from four Spanish vineyards were studied. The specific wine-making technology of these wines requires overripening of the grapes on the vine or extended post-harvest exposure of the grapes in the sun. In every vineyard, samples were taken at three different developmental stages: veraison, harvesting time and after over-ripening. With the maturation of the berries there was a clear increase of Aspergillus spp. In the last sampling time studied, they were isolated from the 90.3% of the plated berries. Black aspergilli (mainly A. niger aggregate and A. carbonarius) were predominant among the different Aspergillus spp. isolated and constituted 98.5% of the total Aspergillus strains isolated. At harvesting time and after over-ripening, the percentage of colonized berries with A. carbonarius exceeded that of Aspergillus niger aggregate. Due to their low frequency of isolation, Penicillium spp. and Aspergillus spp. outside black aspergilli are not an important source of ochratoxin A in grapes for liqueur wine production. On the contrary, 98.5% of the A. carbonarius isolates screened were able to produce ochratoxin A. Although the possible participation of different ochratoxin A-producing species may occur, our results confirm that A. carbonarius is the most important source of ochratoxin A in liqueur wines, increasing its occurrence along the ripening of grapes.     

Australian research on ochratoxigenic fungi and ochratoxin A

The presence of the mycotoxin, ochratoxin A (OTA), has been reported in Australian grape products. Comprehensive surveys of Australian wines have determined that the frequency and level of OTA contamination are low. Aspergillus carbonarius is the primary OTA-producing species associated with grapes in Australia, and all isolates tested to date produce OTA. Aspergillus niger is isolated more frequently from vineyards, however, few strains produce OTA. A. carbonarius and A. niger exist as saprophytes in the top layer of soil beneath vines, from where they are thought to be blown onto bunches. The level of A. carbonarius in soil may be reduced by temperatures above or below the optimum temperature for survival (25 degrees C), by high soil moisture content, and by modifications to tillage and mulching practices. A. carbonarius is an opportunistic pathogen of damaged berries. In the absence of damage, spores may exist on berry surfaces without causing visible rots. Aspergillus rots are associated with black Aspergillus species, primarily A. niger, A. carbonarius and A. aculeatus. The potential for such rots is increased with berry damage, inoculum coverage and berry maturity. Susceptibility to berry splitting is related, in part, to bunch structure, and may be variety-dependent or influenced by rainfall, irrigation and canopy management. Black Aspergillus spp. are closely associated with berries near the main stem of the bunch. During winemaking, around 80% of the OTA initially present in grapes is removed, primarily with the skins and pulp during pressing. Additional reductions occur with the removal of precipitated grape and yeast solids. Bentonite in white wine and yeast hulls in red wine were the most effective non-carbonaceous fining agents for the removal of OTA.     

Toxigenic fungi isolated from dried vine fruits in Argentina

To evaluate the potential for mycotoxin production by fungi in dried vine fruits, the mycobiota was determined both before and after surface disinfection. Predominant genera were Aspergillus (50.2%), Eurotium (21.4%) and Penicillium (13.5%). Aspergillus section Nigri ("black aspergilli") were isolated with relatively high frequency. Aspergillus niger was the most common species but only 3 of 293 isolates screened were ochratoxin A (OTA) producers. Aspergillus carbonarius was less common but 96% of 48 strains screened were ochratoxigenic. OTA was not produced by A. japonicus. Other toxigenic fungi detected were A. ochraceus (3 strains produced OTA), Aspergillus flavus (5 strains produced cyclopiazonic acid but not aflatoxins), P. citrinum (19 strains were strong citrinin producers) and Alternaria alternata (15 strains were producers of tenuazonic acid, alternariol and alternariol methyl ether). In spite of the high incidence of A. carbonarius capable of producing OTA, low levels of this toxin were detected in the samples analysed.     

Ochratoxigenic species from Spanish wine grapes

The ochratoxigenic mycobiota of grapes belonging to representative wine regions located along the Mediterranean coast of Spain at different developmental stages was identified. During the development of the berries, the occurrence of Aspergillus spp. increased while the percentage of berries contaminated by non-ochratoxin A (OTA) producing species such as Alternaria spp. and Cladosporium spp. decreased. Penicillium verrucosum, the only confirmed Penicillium spp. that is able to produce OTA, was not isolated. The contamination by OTA-producing species comes from the surface of the berries and not from the inner fruit. Black aspergilli were predominant among the different Aspergillus spp. isolated. All the Aspergillus carbonarius isolates were able to produce OTA at different concentrations. None of the isolates belonging to Aspergillus niger aggregate and to Aspergillus japonicus var. aculeatus were able to produce OTA. These results are a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine grapes, mainly at the last developmental stages of the berries.     

Effects of fungal interaction on ochratoxin A production by A. carbonarius at different temperatures and a(w)

Ochratoxin A is a well-known mycotoxin produced by species of the genera Penicillium and Aspergillus. OTA-producing species from A. section Nigri are considered the source of OTA detected in grapes, dried vine fruits and wines. Other fungi present in grapes during their maturation can grow and interact with OTA-producing Aspergillus species and affect OTA production. In this study seven fungi (Alternaria alternata, Cladosporium herbarum, Eurotium amstelodami, Trichoderma harzianum, Penicillium decumbens, P. janthinellum and Candida sp.) disolated from grapes and dried vine fruits were grown in SNM medium paired with OTA-positive A. carbonarius at two temperatures (20 and 30 degrees C) and at two water activities (0.92 and 0.97). OTA production was tested after 5, 7, 10, 14 and 18 days of incubation, at four distances (1, 2, 3 and 4 cm) from A. carbonarius inoculation point in the inter-colony axis. At 0.92 a(w) OTA production was almost negligible. At 0.97 a(w) and 30 degrees C OTA accumulation was reduced when A. carbonarius was grown in paired cultures, particularly with A. alternata, C. herbarum, P. decumbens and P. janthinellum. At 0.97 a(w) and 20 degrees C, there was no clear effect of the interacting species on OTA accumulation; in general E. amstelodami and Candida sp. seemed to stimulate OTA production, whereas T. harzianum and P. decumbens reduced it. Competing mycoflora acted as an additional control factor against OTA accumulation at 30 degrees C; but at 20 degrees C, where OTA production is optimal, this did not happen. Thus maintaining the temperature of grapes at or above 30 degrees C during dehydration may provide some control against OTA accumulation in grapes.     

Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market

Ochratoxin A (OTA) can occur in a wide range of foods, but unexpectedly high concentrations have been detected in dried vine fruits of various origins. The European Union has recently established a maximum OTA limit of 10 microg/kg for these foodstuffs. In order to determine the likely origin of OTA, a mycological study of 50 dried fruit samples (currants, raisins, and sultanas) representative of the Spanish market was conducted. Fungal contamination was detected in 49 of 50 (98%) samples. Black aspergilli were isolated from all of the positive samples. Aspergillus niger var. niger was isolated from 98% of the samples, and Aspergillus carbonarius was found in 58% of the samples. One hundred sixty-eight A. niger var. niger isolates and 91 A. carbonarius isolates were screened for their ability to produce OTA. Eighty-eight (96.7%) A. carbonarius isolates and one (0.6%) A. niger var. niger isolate were found to be OTA producers. Black aspergilli were the dominant fungi. Among black aspergilli, A. carbonarius has shown a consistent ability to produce OTA and is the most probable source of this mycotoxin in these substrates.     

Occurrence of ochratoxin A producing fungi in wine and table grapes in Israel

A 3-year survey was conducted to assay the number of Aspergillus Section Nigri isolates and in vitro ochratoxin A (OTA) production capacity in 10 vineyards in Israel. The survey included field sampling of two wine cultivars, 'Sauvignon Blanc' and 'Cabernet Sauvignon' as well as the table grape cultivar 'Superior'. A total of 2114 isolates were analyzed and of those 161 isolates were shown to produce OTA. The major finding was that Aspergillus carbonarius (336 tested strains) is the most consistent producer of OTA, with approximately 35% of the isolates identified as positive in vitro. In comparison, 3.1% of other isolates from the Aspergillus niger aggregate (of 1432 strains) produced OTA in vitro. In contrast, none of the 346 tested strains with a uniseriate head morphology produced OTA. The incidence of infected berries was very low before veraison, while at harvest, this frequency was twice as high. In general, the composition of black Aspergilli did not differ during berry development. Generally, more OTA-producing isolates were isolated from the surface of table grapes cv. 'Superior' compared to 'Sauvignon Blanc'. None of the samples collected at harvest contained traces of OTA in the juice. This study shows that grapes in Israel are contaminated with ochratoxigenic species which represent a risk of OTA contamination.     

Fungi and mycotoxins in vineyards and grape products

Many fungi may occur on grapes during growth in the vineyard, but the main concern from the viewpoint of mycotoxin contamination is the black Aspergilli, Aspergillus carbonarius and A. niger. These fungi are capable of producing ochratoxin A (OA) which may contaminate grapes and grape products such as wine, grape juice and dried vine fruit. Understanding the ecology and physiology of the black Aspergilli can provide tools for management of OA at all stages of grape production and processing. In the vineyard, careful management of cultivation, irrigation and pruning can assist in minimising the levels of black Aspergilli in the soil, which in turn, can minimise contamination of grapes by these fungi. Minimising damage to grapes on the vine by the use of open vine canopies, grape varieties with resistance to rain damage and by the management of insect pests and fungal diseases (e.g., mildew, Botrytis bunch rot) can reduce the incidence of Aspergillus rot in mature berries. The risk of OA in table grapes can be minimised by careful visual inspection to avoid damaged and discoloured berries. In wine, harvesting grapes with minimal damage, rapid processing and good sanitation practices in the winery assist in minimising OA. During vinification, pressing of grapes, and clarification steps which remove grape solids, grape proteins and spent yeast can also remove a significant proportion of OA. For dried vine fruit production, avoiding berry damage, rapid drying, and final cleaning and sorting to remove dark berries can reduce overall OA levels in finished products.     

Detection of ochratoxin A in tropical wine and grape juice from Brazil

BACKGROUND: Ochratoxin A (OTA) is the main mycotoxin found in grapes, wines and grape juices and is considered one of the most harmful contaminants to human health. In this study, samples of tropical wines and grape juices from different grape varieties grown in Brazil were analysed for their OTA content by high‐performance liquid chromatography. RESULTS: The detection and quantification limits for OTA were 0.01 and 0.03 µg L^?1 respectively. OTA was detected in 13 (38.24%) of the samples analysed, with concentrations ranging from < 0.03 to 0.62 µg L^?1. OTA was not detected in any of the grape juice samples. Most of the red wine samples proved to be contaminated with OTA (75%), while only one white wine sample was contaminated. However, the OTA levels detected in all samples were well below the maximum tolerable limit (2 µg L^?1) in wine and grape juice established by the European Community and Brazilian legislature. CONCLUSION: The results of this study indicate a low risk of exposure to OTA by consumption of tropical wines and grape juices from Brazil. © 2012 Society of Chemical Industry     

Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes

Aspergillus carbonarius is the main species responsible for the production of ochratoxin A (OTA) in wine grapes. To monitor and quantify A. carbonarious in grapes, a quantitative real-time PCR assay was developed as a possible tool for predicting the potential ochratoxigenic risk. DNA extraction from grape berries was performed by using conventional extraction and clean up through EZNA Hi-bond spin columns. A TaqMan probe was used to quantify A. carbonarius genomic DNA in grape berries samples. An exogenous internal positive control was used to overcome DNA recovery losses due to matrix inhibition. The quantification of fungal genomic DNA in naturally contaminated grape was performed using the TaqMan signal versus spectrophotometrically measured DNA quantities (Log10) calibration curve with a linearity range from 50 to 5 x 10(-4) ng of DNA. A positive correlation (R2=0.92) was found between A. carbonarious DNA content and OTA concentration in naturally contaminated grape samples. This is the first application of TaqMan real-time PCR for identifying and quantifying A. carbonarius genomic DNA occurring in grapes. The rapid DNA extraction method for grapes, together with the commercial availability of reagents and instrumentation, allows to perform a remarkable number of reproducible assays (96-well format) in less than 4 h.     

Ochratoxin A and ochratoxigenic Aspergillus species in Argentinean wine grapes cultivated under organic and non-organic systems

The evolution of contamination with Aspergillus section Nigri and ochratoxin A occurrence was evaluated in four vineyards located at Mendoza province, Argentina during 2003-2004. The survey included two grape varieties, one of late maturation (Bonarda) and the other of early maturation (Tempranillo). The vineyards were set under non-organic and organic cropping systems. Bunches of grapes at different growth stages were collected, and berries (50 by sample) were plated on Petri dishes containing Dichloran 18% Glycerol Agar (DG18) and Dichloran Rose Bengal Chloramphenicol Agar (DRBC) media. After an incubation period of 7 days at 25 degrees C+/-1 degrees C, the mycoflora belonging to Aspergillus section Nigri was identified. The ability to produce ochratoxin A (OTA) by the potential ochratoxigenic species was evaluated on YES (2% yeast extract, 15% sucrose) medium. The cultures were incubated at 30 degrees C+/-1 degrees C for 10 days in darkness. The OTA content of the grapes was determined by HPLC. Through the different growth stages, from setting to harvest, grape contamination by the Aspergillus species, section Nigri increased. The main species isolated belonged to the A. niger aggregate. From 246 strains evaluated 24% was ochratoxigenic. OTA was not detected in grapes during the survey.     

Aspergillus spp., distribution, population composition and ochratoxin A production in wine producing vineyards in Greece

Vineyard surveys of 11 wine producing grape cultivars, were carried out in sixteen vineyards, in five winemaking regions in Greece, during 2002 and 2003. The occurrence of various Aspergillus spp. in bunches of berries at setting, veraison and ripening at harvest time was investigated. Aspergillus niger aggregate and A. carbonarius were predominantly isolated from sampled berries. Although the prevailing Aspergillus spp. isolates belonged to A. niger aggregate, isolates of A. carbonarius were the most efficient ochratoxin A (OTA) producers. Of 50 tested isolates of A. carbonarius 42% produced amounts of OTA, exceeding 25 ppb, while none of the 85 isolates of A. niger aggregate tested produced above 16 ppb.     

Effect of intra and interspecific interaction on OTA production by A. section Nigri in grapes during dehydration

The aim of this study was to assess the colonizing capability and OTA production of different populations of Aspergillus section Nigri spp. in grapes, as affected by the interactions with other fungi, during a simulated in vitro sun-drying. Mature white grapes were divided into two lots of healthy and artificially injured grapes and inoculated with A. section Nigri spp. (A. carbonarius OTA producer, A. niger aggregate OTA producer, A. niger aggregate OTA non-producer), Eurotium amstelodami and Penicillium janthinellum, in different combinations. The drying process was simulated adjusting water activity firstly at 0.98 a(w) and gradually decreasing it to 0.76 a(w) for a total of 20 days. Colonizing grape percentages were recorded after 5, 10, 15 and 20 days of dehydration and OTA content was measured after 5, 7, 10, 12, 15, 17 and 20 days. Colonization of grapes increased with time in all treatments. A. niger aggregate OTA-positive showed the highest colonization percentage, followed by A. carbonarius, and finally their mixed inoculum. When the two OTA-producing strains were combined, addition of any other microorganism increased the percentage of infection by A. section Nigri. A. carbonarius was the highest OTA producer in pure culture, followed by A. niger aggregate OTA-positive. In general, when competing fungi were added to A. carbonarius inoculum, the OTA content was reduced. E. amstelodami was the only competing fungus which increased OTA accumulation. The sun-drying process may be conducive to OTA accumulation in dried grapes. The complex fungal interactions which may take place during this process, may act as an additional control factor, given that the higher presence of A. niger aggregate OTA-negative inhibits OTA accumulation by OTA producing species.     

What is the source of ochratoxin A in wine?

During a microvinification trial using natural mouldy grapes from a research experimental vineyard, ochratoxin A (OTA) contaminated white wine was obtained. Potential OTA-producing mycobiota of grape samples used in this microvinification process was assessed. Only Aspergillus carbonarius isolates were detected as producers of OTA. Our report is a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine.     

An ITS-RFLP method to identify black Aspergillus isolates responsible for OTA contamination in grapes and wine

Ochratoxigenic mycobiota in grapes from representative wine regions in Valencia was identified. Black aspergilli were predominant among the different Aspergillus spp. isolated. Restriction digestion analysis of the ITS products was tested as a rapid method to identify isolates of black Aspergillus species from grapes. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, NlaIII and RsaI, distinguished five types of restriction fragment length polymorphism (RFLP) corresponding to Aspergillus niger, Aspergillus tubingensis, Aspergillus carbonarius and Aspergillus aculeatus species. In addition, a new RFLP type in the A. niger aggregate was identified. The fragments obtained by digestion with the endonuclease NlaIII could be used to identify these new isolates. Black Aspergillus isolates were tested for their ability to produce OTA. Most of the isolates that produced ochratoxin A in YES medium belonged to A. carbonarius species. These results support evidence that A. carbonarious greatly contributes to OTA contamination in grapes and consequently in wine. The ITS-RFLP assay is proposed as a rapid and easy method to identify black Aspergillus species isolated from grapes, especially in studies that involve a large number of isolates.     

Ochratoxin A occurrence and formation in Portuguese wine grapes at various stages of maturation

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.     

An overview of ochratoxin A in beer and wine

Ochratoxin A (OTA) is a mycotoxin produced mainly by several fungal species of the genera Aspergillus and Penicillium. This mycotoxin has been shown to be nephrotoxic, hepatotoxic, teratogenic and carcinogenic to animals and has been classified as a possible carcinogen to humans. OTA occurs in a variety of foods, including beer and wine. Reports on OTA occurrence in beer indicate that this is a worldwide problem due to the widespread consumption of this beverage. At present, the European Union (EU) has not set a maximum allowable limit (MAL) for this mycotoxin in beer, although there is a limit in barley and malt. Studies carried out in different countries agree in the high proportion of samples contaminated with OTA although levels are, usually, below 0.2 ng/ml. OTA occurrence has been related to the contamination of malt barley with ochratoxigenic species, particularly Penicillium verrucosum. OTA produced in grains is carried to wort and, although fermentation decreases the concentration, the toxin is not eliminated. Reducing the fungal contamination of malt barley is the most promising strategy for reducing OTA in beer. With regard to wine, surveys on the presence of OTA have been conducted worldwide. The proportion of wines in which OTA is detected is very high (above 50%) in some countries (especially in the Mediterranean basin) although only a few wines contained concentrations exceeding the MAL laid down by the EU (2.0 ng/ml). A gradient of concentration is usually recognized; OTA levels decrease in the order red, rose, and white wine but also with increasing latitude of the producing countries. OTA presence in wines is due to the black aspergilli, mainly A. carbonarius, which can grow on grapes in the vineyards and produce the toxin. At grape crushing, the juice can be contaminated with the toxin which is carried over into wine, where it persists due to its stability. Pre- and post-harvest treatments are being investigated to diminish contamination of wines as much as possible.     

Occurrence of fruit rot fungi (Aspergillus section Nigri) on some drying varieties of irrigated grapes

The occurrence of Aspergillus section Nigri on certain varieties of grapes used for dried vine fruit (and especially on Sultana), was surveyed extensively during three harvest seasons (1998, 1999 and 2000). Members of Aspergillus section Nigri were enumerated and identified in a total of 806 samples comprising both fresh fruit as well as partially-dried and fully-dried grapes. Aspergillus aculeatus , A. carbonarius and A. niger were commonly isolated. Of those three species, A. niger showed the highest optimum temperature for growth, as well as highest thermal tolerance. That combination of properties probably accounts for the occurrence of A. niger in over 80% of fruit samples in all three seasons. A. carbonarius was also prevalent on fruit surveyed for fungal rots. Severity of infection with this group of fungi was highest in the seasons when grape berries were rain-damaged. Typically, the Aspergillus count increased during the initial stages of drying. Growth of these moulds while grapes are either still on the vine or during drying carries important implications for human consumption due to the potential for certain strains of these fungi to produce ochratoxin A. Fungal cultures on coconut cream agar plates were screened for production of ochratoxin A via fluorescence emission under UV light. Such fluorescence was observed in all isolates of A. carbonarius , but was not observed in any isolates of either A. aculeatus or A. niger .