Occurrence of ochratoxin A-producing fungi in grapes grown in Italy

A study was carried out to investigate fungi present on grapes grown in Italy. Aspergillus and Penicillium spp. isolates were identified and studied in vitro, and their ability to produce ochratoxin A (OA) was investigated. The survey involved nine vineyards, three located in northern Italy and six located in southern Italy. In 1999 and 2000, bunches of grapes at different growth stages were collected from all nine vineyards, and berry samples were placed in moist chambers and incubated. The resultant fungal colonies were then transferred to petri dishes containing Czapek yeast agar and incubated at 25 degrees C for 7 days; the fungal isolates were identified and then cultivated in liquid Czapek yeast medium and evaluated for their ability to produce OA. During the survey, 508 isolates were collected, with 477 belonging to Aspergillus spp. and 31 belonging to Penicillium spp. Among the aspergilli, species of the Fumigati, Circumdati, and Nigri sections were identified, with species of the Nigri section (464 isolates) largely predominating; for species of the Nigri section, 108 isolates were uniseriate, 270 were biseriate, and 86 were identified as Aspergillus carbonarius. Black aspergilli isolated over the 2 years of the study showed a very similar pattern. On average, the biseriates represented about 60% of the isolates collected in both years and were followed by uniseriates (21%) and A. carbonarius (19%). The most toxigenic strains proved to be those of A. carbonarius; about 60% of these isolates were OA producers and produced the highest levels of OA. A. carbonarius was more frequent in the south, but in both areas the percentages of OA-producing isolates remained the same.     

Study of ochratoxin A-producing strains in coffee processing

Ochratoxin A (OTA) is the main mycotoxin that has been detected in coffee. The occurrence of OTA in coffee beans can be because of environmental conditions and/or processing conditions. Three coffee processes were evaluated (wet, mechanical and dry processes), at different stages from harvesting to storage, and fungi producing OTA were enumerated and identified. The frequency of potential OTA‐producing fungi and their ability to produce OTA was also studied. By direct plating, the levels of contamination found in the coffee processes were 80, 72 and 92%, respectively, for parchment and dry cherry coffee and 20, 34 and 15% for green coffee. Aspergillus ochraceus isolated from the three processes accounted for 6.6, 8.3 and 3.3%, and Aspergillus niger for 15, 13 and 25% of the strains isolated, respectively. The toxigenic potential of five A. ochraceus and two A. niger strains was tested in FDA medium and coffee medium using the HPLC technique. There was no difference between the processes studied in terms of isolation and occurrence of ochratoxigenic fungi.     

Advances in the molecular diagnosis of ochratoxin A-producing fungi

Ochratoxin A (OTA) is detected worldwide in various food and feed sources. The compound is produced by Penicillium nordicum and P. verrucosum, as well as by various species within the sections Nigri and Circumdati of the genus Aspergillus, with A. ochraceus and A. carbonarius known to be the predominant producers. Recently, various pairs of PCR primers based on AFLP, RFLP, RAPD and the calmodulin gene were developed to set up novel diagnostic approaches for OTA producers in the Aspergillus and Penicillium genera. Real-time PCR assays based on well-characterized genomic sequences in A. ochraceus and P. nordicum have also been set up. Since the application of such assays to the analysis of contaminated sample material was demonstrated in only a few cases, future studies should be focused on applying such methods in rapid, robust and user-friendly applications, and implementing them in HACCP concepts. The recent detection and characterization of OTA biosynthetic pathway genes in the Penicillium genus is an important step towards understanding what mechanisms influence production of the toxin in order to redesign production processes in the food and feed industry and to keep de-novo synthesis to a minimum.     

Studies on the sample weight for ochratoxin A determination in raw coffee

Ochratoxin A is a mycotoxin that may cause damage to the kidneys and the immune system in man. Foods are examined for this contaminant by the laboratories responsible for official food control. At present, there are no specific provisions for the sampling and examination of ochratoxin A in foods. Therefore, recourse is made to the provisions for aflatoxins. They prescribe the processing of samples with a weight of up to 30 kg. The examination of raw coffee for ochratoxin A in conjunction with the German Food Monitoring Programme of 2000 was undertaken with various sample weights (5, 10 and 30 kg) in order to identify the influence of the sample weight on the result. Overall, the sample weight did not have an effect on the detection and level of the ochratoxin A content in the samples of raw coffee examined.     

Occurrence of toxigenic fungi in ochratoxin A contaminated liquorice root

Fungi associated with ochratoxin A (OTA)-contaminated liquorice root and their capabilities for OTA production were investigated. Medicinal materials of mouldy liquorice root were collected from herbal markets located in Jiangxi, Zhejiang, Henan provinces and Beijing, China, respectively. Sixteen fungal species belonging to Penicillium, Aspergillus, Eurotium, Fusarium, Mucor and Scopulariopsis were isolated; the fungal composition was different in each liquorice root sample. Penicillium polonicum was predominant, comprising 54% of the total isolates in the liquorice root sample from Jiangxi province, which was contaminated with OTA at the highest level. In other samples with lower OTA contents, species of Aspergillus and Eurotium were predominant. OTA production of representative strains on rice media was detected by LC-MS/MS; all Penicillium polonicum isolates and a P. chrysogenum were ochratoxigenic; OTA concentrations ranged from 6.94 to 217.37?ng?g^?1. This is the first study to report P. polonicum as an OTA-producing fungus. OTA contamination of mouldy liquorice root constitutes a major health hazard in consumption. This situation demands urgent and undivided attention.     

Occurrence of toxigenic fungi in ochratoxin A contaminated liquorice root

Fungi associated with ochratoxin A (OTA)-contaminated liquorice root and their capabilities for OTA production were investigated. Medicinal materials of mouldy liquorice root were collected from herbal markets located in Jiangxi, Zhejiang, Henan provinces and Beijing, China, respectively. Sixteen fungal species belonging to Penicillium, Aspergillus, Eurotium, Fusarium, Mucor and Scopulariopsis were isolated; the fungal composition was different in each liquorice root sample. Penicillium polonicum was predominant, comprising 54% of the total isolates in the liquorice root sample from Jiangxi province, which was contaminated with OTA at the highest level. In other samples with lower OTA contents, species of Aspergillus and Eurotium were predominant. OTA production of representative strains on rice media was detected by LC-MS/MS; all Penicillium polonicum isolates and a P. chrysogenum were ochratoxigenic; OTA concentrations ranged from 6.94 to 217.37?ng?g(-1). This is the first study to report P. polonicum as an OTA-producing fungus. OTA contamination of mouldy liquorice root constitutes a major health hazard in consumption. This situation demands urgent and undivided attention.     

Current importance of ochratoxin A-producing Aspergillus spp

Ochratoxin A (OA) is receiving attention worldwide because of the hazard it poses to human and animal health. OA contamination of commodities, such as cereals or pork and poultry meat, is well recognized. Nevertheless, there is an increasing number of articles reporting OA contamination in other food commodities, such as coffee, beer, wine, grape juice, and milk, in the last few years. This continuous and increasing exposure to OA that humans experience is reflected in the high incidence of OA in both human blood and milk in several countries. OA was believed to be produced only by Aspergillus ochraceus and closely related species of section Circumdati and by Penicillium verrucosum; however, in the genus Aspergillus, the production of OA has been recently reported by species outside the section Circumdati. Thus, it has been clearly established as a metabolite of different species of the section Nigri, such as Aspergillus niger and Aspergillus carbonarius. OA production ability by Aspergillus spp. is more widespread than previously thought; therefore, there is the possibility that unexpected species can be new sources of this mycotoxin in their natural substrates.     

Isolation, identification and toxigenic potential of ochratoxin A-producing Aspergillus species from coffee beans grown in two regions of Thailand

In 2006 and 2007, 32 Thai dried coffee bean samples (Coffea arabica) from two growing sites of Chiang Mai Province, and 32 Thai dried coffee bean samples (Coffea canephora var. robusta) from two growing sites of Chumphon Province, Thailand, were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The overall percentage of fungal contamination in coffee was 98% and reduced to 60% after surface disinfection. There were remarkable ecological differences in the composition of ochratoxigenic species present in these two regions. Arabica coffee bean samples from the North had an average of 78% incidence of colonization with Aspergillus of section Circumdati with Aspergillus westerdijkiae and A. melleus as the predominant species. Aspergillus spp. of section Nigri were found in 75% of the samples whereas A. ochraceus was not detected. Robusta coffee beans from the South were 98-100% contaminated with predominantly A. carbonarius and A. niger. A. westerdijkiae was only found in one sample. The diversity of the fungal population was probably correlated with the geographical origin of the coffee, coffee cultivar, and processing method. Representative isolates of section Circumdati (52) and Nigri (82) were examined for their OTA production using HPLC with fluorescence detection. Aspergillus westerdijkiae (42 isolates out of 42), A. steynii (13/13), and A. carbonarius (35/35) in general produced large amounts of OTA, while one isolate of A. sclerotiorum produced intermediate amounts of OTA. 13% of the A. niger isolates produced OTA in intermediate amounts. OTA levels in coffee bean samples were analyzed using the Ridascreen OTA ELISA kits. Of the 64 coffee bean samples analyzed, 98% were contaminated with OTA in levels of <0.6-5.5 microg/kg (Arabica) and 1-27 microg/kg (Robusta). Presence of OTA in representative coffee samples was also confirmed by LC-MS/MS after ion-exchange purification.     

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 source of ochratoxin A in Brazilian coffee and its formation in relation to processing methods

A total of 408 Brazilian coffee samples was examined during the 1999 and 2000 coffee harvest seasons for the presence of ochratoxin A (OA) and fungi with the potential to produce it. Samples came from four regions: Alta Paulista (western area of S?o Paulo State), Sorocabana (southwest S?o Paulo State), Alta Mogiana (northeast S?o Paulo State) and Cerrado Mineiro (western area of Minas Gerais State). Cherries and beans were examined at different stages: immature, mature and overripe cherries from trees, overripe cherries from the ground and beans during drying and storage on the farm. For mycological studies, the cherries and beans were surface disinfected with chlorine, plated on Dichloran 18% Glycerol Agar at 25 degrees C for 5-7 days and analysed for the presence of Aspergillus ochraceus and closely related species, A. carbonarius and A. niger. More than 800 isolates of fungi belonging to these species were identified and studied for the ability to produce OA using the agar plug technique and thin layer chromatography (TLC). A. niger was the species found most commonly (63% of isolates of these three species), but only 3% of them produced OA. A. ochraceus also occurred commonly (31% of isolates), and 75% of those studied were capable of OA production, a much higher percentage than reported elsewhere. A. carbonarius was found (6% of isolates) only in Alta Paulista, the hottest region studied, and only from beans in the drying yard or in storage. However, 77% of the A. carbonarius isolates were capable of producing OA. Average infection rates for cherries taken from trees were very low, but were higher in fruit taken from the ground, from the drying yard and from storage, indicating infection by toxigenic species after harvest. The average OA content in 135 samples of mature cherries from trees, overripe from trees, overripe from the ground, drying yard and storage was 0.1, <0.2, 1.6, 2.1 and 3.3 microg/kg, respectively. Although individual OA levels varied widely, only 9 of the 135 samples analysed exceeded 5 microg/kg OA, with one sample of poor quality dried coffee in excess of 100 microg/kg OA. The causes of high contamination were investigated on the farms concerned and several critical points were found, relating both to local climatic conditions and the drying processes used.     

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.     

葡萄上赭曲霉毒素A产生机制及生物检测方法研究进展

赭曲霉毒素A(Ochratoxin A,OTA)主要由赭曲霉、黑曲霉及青霉菌的某些菌株产生,具有多种毒性,葡萄及其制品中OTA污染在全球范围内普遍存在,严重影响人类和动植物的健康。控制OTA的方法已经有许多研究,其中生物方法安全环保是目前研究的热点,而认识OTA来源及产生机制是生物防治污染的基础,随着分子生物学的发展,一些新型的分子快速检测方法已经被研究出来。对OTA的毒理特性、污染及来源、产毒机制、分子生物学快速检测手段等方面进行了综述,为寻求有效的控制产毒和降解毒素方法提供理论参考,以期降低OTA污染。     

Occurrence of phenols in coffee melanoidins

Compounds with higher molecular weights in roast coffee were separated by means of adsorption chromatography followed by gel chromatography which yielded seven fractions of different molecular weights. These were tested sensorially and degraded by Curie point pyrolysis high-resolution gas chromatography/mass spectrometry (HRGC/MS) about 100 products, among them 33 phenols, were found. The products were compared with fragments formed via model pyrolysis experiments on chlorogenic acid.     

Occurrence of ochratoxin A in Australian wine

A survey of 601 samples of Australian wine for the presence of ochratoxin A (OA) was undertaken. The study sampled predominantly bottled wines, approximately equal numbers of red and white wines, wines made from a range of grape varieties and wines from all the major producing areas and some of the minor ones. A validated HPLC assay that did not utilise expensive immunoaffinity columns was developed for quantifying OA in wine, with limits of detection and quantification of 0.02 and 0.05 μg/L respectively. OA was detected in 90 (15.0%) of the 601 samples, but at uniformly low levels, with 85% of positive samples containing less than 0.2 μg/L OA. Only one sample exceeded 0.5 μg/L OA and no sample exceeded 1.0 μg/L OA. No region of Australia appeared more or less likely to produce wines containing OA.     

Occurrence of ochratoxin A in Greek wines

The presence of ochratoxin A in foods and wines is of current interest owing to its toxic effects. Ochratoxin A is a widely distributed mycotoxin with nephrotoxic activity. It constitutes a new restrictive agent for the export of vinicultural products and is thus important from an economic as well as a scientific point of view. In this study the determination of ochratoxin A in Greek wines was made by means of commercial immunoaffinity columns for purification followed by HPLC with fluorescence detection for quantification of the toxin. The method was applied to 28 dry wines (14 red, 13 white, one rosé) and seven sweet wines (three red, four white) obtained by various viticultural and oenological practices. The levels of ochratoxin A ranged from <0.02 to 3.2 ng ml^?1, with sweet wines containing higher levels than dry wines. Most of the wines contained relatively low concentrations of ochratoxin A (0.02–0.5 ng ml^?1), which do not represent a serious risk to consumer health. © 2003 Society of Chemical Industry     

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.     

A polyphasic approach to the identification of ochratoxin A-producing black Aspergillus isolates from vineyards in Sicily

Aspergillus strains belonging to section Nigri isolated during a two year survey in eight Sicilian vineyards located on the slopes of Mount Etna (Sicily, Italy) were analysed analyzed in order to characterize species responsible for ochratoxin A (OTA) contamination of grapes. The polyphasic approach permitted analysis of biodiversity of Aspergillus isolates in relation to their morphology, ochratoxigenicity and genetic variability. We assessed OTA production by A. carbonarius, A. niger, A. tubingensis and A. japonicus using an enzyme-linked immunosorbent assay. A. carbonarius isolates were the strongest OTA producers. A subset of 66 representative strains was selected for further DNA-based characterization. PCR assays using species-specific primers discriminated between A. niger, A. carbonarius and A. japonicus on the basis of the target sequences for each species. The PCR-based methods matched morphological characterization in identifying all the black aspergilli (BA) isolates tested, whereas RFLP analysis with RsaI of isolates positive to PCRs with A. niger specific primers identified three A. tubingensis isolates. The identification of thirteen isolates was further confirmed by ITS analysis. By this method, each of the isolates was identified and assigned to an Aspergillus species. The fAFLP analysis of 40 isolates highlighted the power of this technique to discriminate different species and single strains, to verify the presence of mixed populations in the same vineyard, through homogeneous species clusters. No correlation was observed between the clusters and OTA production level or origin.     

Occurrence of ochratoxin A in raw ham muscle,salami and dry-cured ham from pigs fed with contaminated diet

Pork meat-derived products can contribute to the overall ochratoxin A intake, either by carry-over effect, or by environmental mould population cross-contamination. In order to assess the role of these different contamination routes, a study was carried out with pigs challenged orally with OTA contaminated feed at subchronical level. After slaughtering, thighs and minced meat from control and treated groups were transformed into dry-cured hams and salami, respectively, which were analysed for OTA determination after ripening. From collected data, the carry-over in muscle was generally low, whereas a significant contribution to the OTA contamination in dry-cured hams was due to toxinogenic mould population growing on their surface during ripening. Finally, a survey of different types of dry-cured ham (n = 110), from the Italian market, was performed, showing the occurrence of OTA on the surface portion in 84 out of 110 samples with a median value of 0.53 μg/kg and in the inner core in 32 out of 110 samples with a median value lower than 0.1 μg/kg.     

Occurrence of ochratoxin A in Rioja Alavesa wines

In this survey the influence of the geographical location and the kind of wine in the concentration of ochratoxin A (OTA) was studied. Forty percent of the Spanish wine market belongs to the Rioja Qualified Designation of Origen (DOCa Rioja) wines, which are already worldwide known. A total of 100 wines from the Rioja Alavesa (RA) production area of the DOCa Rioja were analysed, using immunoaffinity column clean-up and high-performance liquid-chromatography with fluorimetric detection (detection limit 0.002 μg/l). The presence of OTA was greater in wines produced in low rainfall and high temperature regions. The geographical location and kind of wine did not seem to have influence on the OTA concentration.