Occurrence of ochratoxin A in wine and ochratoxigenic mycoflora in grapes and dried vine fruits in South America

Grape and wine production in South America represents about 6.6% and 10% respectively of the world grape and wine production. The available information on the ochratoxigenic mycoflora and ochratoxin A (OTA) presence in wine grapes, wines, grape juices and dried vine fruits is limited. Surveys have been carried out in Argentina and Brazil which showed that Aspergillus niger aggregate are predominant in the Argentinean varieties while from the Brazilian varieties the species A. niger, Aspergillus ochraceus and Aspergillus carbonarius were isolated. A mycobiota survey from wine grapes in Argentina showed that while Alternaria alternata was predominant, Aspergillus section Nigri species were isolated from 60% of samples. About 41% of black Aspergilli isolates produced OTA with levels ranging from 2 to 24.5 ng mL(-1). In another study, about 83% of A. carbonarius isolates from dried vine fruits produced OTA, with levels ranging from 2 to 5200 ng mL(-1). A survey of grape juices and wines of Brazilian, Argentinean and Chilean origin were found to contain very low levels of OTA. Studies are in progress in Latin America on the ecophysiology of ochratoxigenic fungi and OTA occurrence to reduce the impact of this toxin in the food chain.     

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.     

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 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.     

AFLP characterization of Southern Europe population of Aspergillus Section Nigri from grapes

Members of Aspergillus belonging to Section Nigri are distributed worldwide and are mainly responsible for the ochratoxin A accumulation in grapes and wine, particularly in Southern Europe. Limited information is available on the species composition and genetic variability of black Aspergilli strains occurring on grapes. We analyzed 283 representative strains from the main wine producing European countries collected in 2001-2002 (Italy, France, Spain, Portugal, Greece and Israel) using amplified fragment length polymorphisms (AFLP) technique. Four main groups were obtained by AFLP clustering analysis of these strains and three of them showed a well defined homogeneous population/species with intraspecific homology higher than 48%: Aspergillus carbonarius (105 strains), Aspergillus tubingensis (69 strains), and Aspergillus "uniseriate" (56 strains) with a similarity less than 20% to the Aspergillus japonicus type strain. The fourth cluster, that we called "A. niger like" (44 strains), showed low homology with A. niger type strain (35%) and high internal heterogeneity. Finally, nine strains could not be assigned readily to any of the type strain of the A. nigri Section. These findings indicate that the Aspergillus Section Nigri strains occurring on grapes in Southern Europe represent a complex of species, and some of these are peculiar to grapes.     

RFLP characterization of Aspergillus niger aggregate species from grapes from Europe and Israel

In order to characterize by molecular methods the Aspergillus niger aggregate species involved in the ochratoxin A (OTA) contamination of European wine grapes and table grapes from Israel, a total of 173 strains were studied. The ITS-5.8S rDNA fragments of 173 A. niger agreggate strains from grapes included in this study were amplified and their PCR amplicons were RsaI digested in order to classify the strains in the RFLP types, N and T. All of the strains belonging to the A. niger aggregate were classified into the two RFLP types previously defined: type N (43%) and type T (57%). Twenty out of the 173 strains of A. niger aggregate produced OTA (0.1 to 10.5 mug g(-1)). All the OTA producing species belonged to the N-RFLP type.     

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 .     

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.     

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.     

Incidence of fumonisin B2 production within Aspergillus section Nigri populations isolated from California raisins

Fungi belonging to Aspergillus section Nigri occur frequently and in high populations on grapes. Species within this section include Aspergillus niger, A. tubingensis, and A. carbonarius, and they are potential sources for mycotoxins including ochratoxin A and fumonisin B(2) (FB(2)) in grapes and grape products. Aspergillus section Nigri strains were isolated from California raisins to examine the frequency and extent of FB(2) production. Of 392 strains isolated, 197 strains were identified as A. niger, 131 of which produced FB(2). These strains produced from 1.2 to 27 μg/ml FB(2) in culture. PCR amplification of fum1 and fum19 gene fragments showed that all FB(2)-producing strains and nearly all nonproducing strains of A. niger contain these genes. An additional 175 strains were identified as A. tubingensis, none of which produced FB(2). PCR with fum1 and fum19 primers amplified gene fragments of 14 and 25% of A. tubingensis strains, respectively, suggesting that putative orthologs of A. niger fumonisin biosynthetic genes might occur in A. tubingensis. These results indicate that FB(2) production is common among field isolates of A. niger and suggest that the potential for FB(2) contamination of California raisins should be addressed further.     

Digestion of DNA regions to discriminate ochratoxigenic and non-ochratoxigenic strains in the Aspergillus niger aggregate

Aspergillus strains belonging to the Aspergillus niger aggregate, either isolated from Italian grapes or received from public collections, were analysed in order to discriminate between the ochratoxin A (OTA) producing and the non-producing strains by means of the analysis of Internal Transcribed Spacers (ITS), Intergenic Spacers (IGS) and of a beta-tubulin gene portion. A. niger and Aspergillus awamori were identified observing the macro- and microscopic features of the colonies and the strains ochratoxigenicity was evaluated through Thin Layer Chromatography and/or High Performance Liquid Chromatography. PCR amplification of ITS, IGS and beta-tubulin gene portion produced 600, 440 and 550 bp amplicons, respectively, in all the analysed strains. The digestion of the IGS amplicon with Hinf I and of the other two amplicons with Rsa I, revealed one main profile type ("T-C-C") associated with the A. niger aggregate strains which lack the ability to produce OTA.     

Characterization of filamentous fungi isolated from Moroccan olive and olive cake: toxinogenic potential of Aspergillus strains

During the 2003 and 2004 olive oil production campaigns in Morocco, 136 samples from spoiled olive and olive cake were analyzed and 285 strains were isolated in pure culture. Strains included 167 mesophilic strains belonging to ten genera: Penicillium, Aspergillus, Geotrichum, Mucor, Rhizopus, Trichoderma, Alternaria, Acremonium, Humicola, Ulocladium as well as 118 thermophilic strains isolated in 2003 and 2004, mainly belonging to six species: Aspergillus fumigatus, Paecilomyces variotii, Mucor pusillus, Thermomyces lanuginosus, Humicola grisea, and Thermoascus aurantiacus. Penicillium and Aspergillus, respectively, 32.3 and 26.9% of total isolates represented the majority of mesophilic fungi isolated. When considering total strains (including thermotolerant strains) Aspergillus were the predominant strains isolated; follow-up studies on mycotoxins therefore focused primarily on aflatoxins (AFs) and ochratoxin A (OTA) from the latter strains. All isolated Aspergillus flavus strains (9) and Aspergillus niger strains (36) were studied in order to evaluate their capacity to produce AFs and OTA, respectively, when grown on starch-based culture media. Seven of the nine tested A. flavus strains isolated from olive and olive cake produced AF B1 at concentrations between 48 and 95 microg/kg of dry rice weight. As for the A. niger strains, 27 of the 36 strains produced OTA.     

甘肃河西走廊产区酿酒葡萄产毒霉菌的分离与鉴定

目的：了解甘肃河西走廊酿酒葡萄产区污染霉菌的主要类群,分析酿酒葡萄潜在的霉菌毒素污染。方法：采集河西走廊不同地区的酿酒葡萄,进行霉菌的分离纯化和鉴定,并通过高效液相色谱法测定霉菌代谢产物。结果：鉴定出4 株污染酿酒葡萄的优势菌,分别为青霉属的黄绿青霉（Penicillium decumbens）和扩展青霉（Penicillium expansum）,曲霉属的杂色曲霉（Aspergullus versicolor）和烟曲霉（Aspergillus fumigatus）;扩展青霉能够产生展青霉毒素。结论：根据污染酿酒葡萄霉菌的情况,可确定河西走廊酿酒葡萄产区存在展青霉毒素污染的风险。     

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.     

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.     

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.     

The effects of cold storage of table grapes, sulphur dioxide and ethanol on species of black Aspergillus producing ochratoxin A

Table grapes of cv. 'Superior' were sampled from four vineyards during three seasons, for the occurrence of black Aspergillus species that can produce ochratoxin A after storage. The ochratoxin A-producing fungus, Aspergillus carbonarius , was identified in all the samples at harvest and it survived through cold storage. Storage of table grapes for 7 days at 20 °C resulted in occasional increase in the number of isolates recovered from the berries. In contrast, storage for 1 month at 0 °C, with sulphur dioxide (SO₂) generator pads reduced the number of isolates significantly or completely, depending on the dose. Dipping the clusters in ethanol prior to cold storage did not reduce the number of isolates of the Aspergillus niger aggregate after storage. Exposure of A. carbonarius to a final level of 0.4 ppm of SO₂ resulted in fewer fungal colonies than in the control, and the surviving spores developed into fungal colonies that failed to sporulate. These results demonstrate that A. carbonarius is ubiquitous on table grapes before storage and care should be taken to avoid its contamination.     

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.     

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.     

The occurrence of Staphylococcus aureus in fermented milk products (fura and manshanu) in Nigeria

Ninety-three samples of fermented milk cereal (Fura) and 79 of local butter (Manshanu) were collected from four different markets around Zaria. For Fura the mean content of Staphylococci for each of the four markets ranged from 4.5 × 10³ to 4.3 × 10⁴ cfu/ml and the mean aerobic mesophilic plate count from 5.6 × 10⁵ to 2.7 × 10⁶ cfu/ml. For Manshanu the mean staphylococcal count and aerobic mesophilic plate count ranged from 3.4 × 10² to 2.2 × 10³ cfu/ml and 6.7 × 10⁴ to 1.1 × 10⁶ cfu/ml respectively. Significant differences were seen between the different markets.     

MICROBIOLOGICAL PROFILE OF MURCHA STARTERS AND PHYSICO-CHEMICAL CHARACTERISTICS OF POKO, A RICE BASED TRADITIONAL FERMENTED FOOD PRODUCT OF NEPAL

Poko is a traditional rice based fermented food of Nepal prepared using murcha as the starter. The microbiological evaluation of eleven murcha starters showed that the lactic acid bacteria and yeast were dominant at 5×10⁵ to 1.0×10⁹ cfu g^-1 range while fungi were present at 2×10⁵ to 1.0×10⁷ cfu g^-1. Coliforms (1×10² to 1.4×10⁵ cfu g^-1), E. coli (1×10³ cfu g^-1), and. S. aureus (1×10² g^-1) were present in some of the murcha starters. Bacillus cereus was absent in all the starters. The microbial succession during poko fermentation suggested that it was primarily a mixed fermentation of yeasts, molds and lactic acid bacteria. The quality poko product obtained after two days and three days of fermentation at 30°C had a pH, acidity, reducing sugar, total sugar, and alcohol in the range of 3.2-3.0, 1.1-1.3 (% LA), 14.4-15.6 (%), 14.6-18.2 (%) and 1-1.6 (%) respectively. These critical ranges of biochemicals in the poko, due to the fermentation process imparted desirable organoleptic characteristics to the product. Saccharomyces cerevisiae, Candida versatilis, Lactobacillus spp, Pediococcus spp and Rhizopus spp were the dominant microorganisms identified from the poko fermentation. The results suggest that the quality of poko depends upon the microbial flora of the traditional murcha starters.