Revision of ochratoxin a production capacity by the main species of Aspergillus section Circumdati. Aspergillus steynii revealed as the main risk of OTA contamination

Ochratoxin A (OTA) is a highly toxigenic mycotoxin commonly present in a number of diverse agroproducts. Aspergillus Section Circumdati includes some of the most important OTA-producing species: Aspergillus ochraceus, Aspergillus elegans and the recently described Aspergillus steynii and Aspergillus westerdijkiae. In this work, OTA production by several strains of these four species from diverse origins and food matrices was examined. Identification of all the strains was carried out by specific PCR assays. The strains were cultivated in CYA solid medium and OTA was measured by HLPC. This study demonstrated for the first time the high capacity of A. steynii strains to produce OTA at higher levels than A. westerdijkiae,A. ochraceus and A. elegans strains showed low levels or no production at all. In conclusion, the results of this study suggest that the occurrence of A. steynii and A. westerdijkiae might represent the major potential risk for OTA contamination due to their high production and the diversity of commodities that these species may contaminate.     

Effect of two different roasting techniques on the Ochratoxin A (OTA) reduction in coffee beans (Coffea arabica)

The roasting of green coffee beans (Coffea arabica) artificially contaminated by Ochratoxin A (OTA) after inoculation with Aspergillus westerdijkiae was carried out with two different roasting techniques (Rotating Cylinder [RC] and Fluidized Bed [FB]). The green coffee beans were contaminated at two different toxin levels (L1 = 5.3 μg kg^?1 and L2 = 57.2 μg kg^?1). Different roasting points (light, medium, dark and very dark) were set according to the L¹ color coordinate. The cylinder roasting conditions were 0, 3, 6, 9, 12, and 15 min at 230 °C and the fluidized bed roasting conditions were 0, 0.9, 1.7, 2.6, 3.5 and 4.3 min, at 230 °C. The roasted beans were compared for their physical properties (bean swell and weight loss) as well as for their residual OTA content. The results indicated that the OTA reduction was similar for the two contamination levels: 95.1% and 97.2% with the rotating cylinder and 81.3% and 79.2% with the fluidized bed at the maximal roasting time. The OTA degradation kinetics differed between the two processes. The complete degradation of OTA within the limit of this study (230 °C) was not observed but the rotating cylinder roasting was the most efficient technical process for the OTA reduction in a commercial dark roasted coffee (88%).     

Ochratoxin A in coffee beans (Coffea arabica L.) processed by dry and wet methods

The incidence of ochratoxin A was studied in different coffee (Coffea arabica L.) samples. A higher incidence of filamentous fungi was observed in the coffee swept from ground and floating coffee samples. The species Aspergillus ochraceus, Aspergillus sulphureus and Aspergillus sclerotiorum were ochratoxin A producing. In 128 (44%) samples ochratoxin A was not detected; however, in 89 samples (31%), ochratoxin A was detected at 0.1–5.0 μg/Kg levels. Other 25% samples presented contamination above 5.0 μg/Kg. This study showed that the fractions coffee swept from ground and floating coffee represents a serious risk of ochratoxin A contamination.     

Kinetics of ochratoxin A destruction during coffee roasting

In the present study, Coffea arabica was artificially contaminated with spores of toxigenic Aspergillus westerdijkiae. The contaminated coffee was roasted in a vertical spouted bed roaster at four different temperatures (180 °C, 200 °C, 220 °C and 240 °C) and three different time periods (5, 8 and 12 min), in order to obtain more accurate results for the development of the kinetic model for ochratoxin A (OTA). Chlorogenic acids (CGA) content during coffee roasting was also evaluated to investigate the effect of the heat employed to destroy OTA in these health promoting compounds. Coffee treated with spouted bed roasting significantly reduced the OTA level from 8% to 98%. The spouted bed roasting proved to be a very efficient procedure for OTA reduction in coffee, and its reduction depended directly on the degree of roasting. OTA degradation during coffee roasting followed first order reaction kinetics. Using the apparent activation energy of OTA degradation and the temperature-dependent reaction rate, there was a compliance with the Arrhenius equation. This model was capable of predicting the thermal induced degradation of OTA and may become an important predicting tool in the coffee industry. The present study was also able to propose roasting conditions appropriate to destroy OTA and maintain most of the CGA at the same time.     

Ochratoxin A and ochratoxigenic black Aspergillus species in Tunisian grapes cultivated in different geographic areas

This paper summarizes the results of a large study on the occurrence of ochratoxigenic fungi and Ochratoxin A from wine and table grapes in Tunisia. Our results revealed that Aspergillus section Nigri were the unique potential OTA producing fungi isolated from grapes. Isolates belonging to Aspergillus niger aggregate were the most abundant species followed by Aspergillus carbonarius isolates, then uniseriate aspergilli. A. carbonarius presented the highest percentage of OTA-positive strains (97%) whereas only 3% of A. niger aggregate isolates were OTA positive. Grapes were analysed for their OTA content and 58% of them contained detectable levels of OTA, between 0.05 and 5.85 μg/l. Only 4 samples out of 39 exceeded the OTA limit of 2 μg/l fixed by the EU for wine and grape juices. The most contaminated grapes were those from Raf-Raf region located in the North-Est and characterized by a humid climate. Grapes from the Regueb region, characterized by an arid climate, were rarely contaminated. Furthermore, A. carbonarius, which is the main OTA producer fungi on grapes, was rarely isolated in Regueb.     

Occurrence of Ochratoxin A in coffee beans,ground roasted coffee and soluble coffee and method validation

The purpose of this work was to determine the occurrence of Ochratoxin A (OTA) in coffee beans, ground roasted coffee and soluble coffee, which is imported by Argentina and manufactured in this country and also to perform a single laboratory validation for the analysis of OTA. Validation was done with certified reference material and with spiked samples. Certified material showed 104% of toxin recovery in the case of roasted coffee and 100% for green coffee. Spiked samples with levels from 1.98 to 10.18 μg/kg for soluble coffee had an average recovery rate of 79.4%. The limits of detection and quantification in coffee were 0.02 μg/kg and 0.05 μg/kg respectively. A good correlation (r = 0.9989) was found for this method. Fifty one samples were investigated to determine the presence of OTA, extracted by Ochraprep^® immunoaffinity columns for cleaning up and analysed by high performance liquid chromatography (HPLC). The results showed that a high percentage (69%) of the coffee was contaminated with OTA at different levels. The median obtained for green coffee was 2.7 μg/kg, for ground roasted coffee was 0.24 μg/kg and 0.43 μg/kg for soluble coffee. A possible exposure assessment was evaluated.     

Application of PCR-DGGE to the study of dynamics and biodiversity of yeasts and potentially OTA producing fungi during coffee processing

IntroductionOchratoxin A (OTA) is a toxic secondary metabolite produced by fungi of the genera Aspergillus and Penicillium. It has been shown to have carcinogenic and immunotoxic properties in rats and to be responsible for human and animal kidney pathologies. OTA content in coffee was shown to be closely link to harvesting conditions, postharvest processing conditions and especially dry processing, storage and transportation conditions.PurposeFairly little is known about the conditions for contamination by fungi responsible for OTA production, their propagation and conditions for OTA production. Biodiversity and dynamics of fungal populations linked to OTA production could be studied by PCR-DGGE genetic fingerprinting with the aim to understand the effects of postharvest processing on the microbiota.ResultsDGGE fingerprints analyzed by multivariate analysis showed an evolution of the fungal microflora of coffee during the different stages of the postharvest treatments (wet and dry processes) by extraction and amplification of 26S (yeasts) and 28S rDNA (filamentous fungi). PCR-DGGE stages were optimized: extraction and amplification, repeatability and sensitivity methodology applied to fungi were tested.ConclusionPCR-DGGE is a rapid molecular technique to monitor the dynamics of coffee microbial populations (fungi and yeast).Significance of studyPCR-DGGE is a promising tool in order to investigate OTA production in coffee beans.     

Effect of different roasting levels and particle sizes on ochratoxin A concentration in coffee beans

Contamination of roasted coffee with ochratoxin A (OTA) is directly related to the processing quality throughout the coffee production chain, from the farming to the roasting processes. The aim of this study was to evaluate the effects of roasting and particle size on the residual concentration of ochratoxin A in roasted and ground coffee. Coffee beans were artificially contaminated with Aspergillus ochraceus. The beans were roasted to three levels (light, medium and dark) and ground into three types (fine, medium and coarse) after an incubation period. OTA quantification was performed using high-performance liquid chromatography. The combination of dark roast and coarse particle size had the lowest concentration of OTA, 3.06 μg/kg with a 97.17% reduction. The results of this study show that roasting and particle size, rather than roasting alone, are critical for the residual concentration of OTA in roasted and ground coffee beans.     

Ochratoxin A in Spanish retail ground roasted coffee: Occurrence and assessment of the exposure in Catalonia

Occurrence of ochratoxin A (OTA) in ground roasted coffee from different brands and types available in Spain was assessed. Based on these data, exposure of the Catalan population to OTA through coffee consumption was estimated. Coffee samples were purchased in hypermarkets and supermarkets of twelve cities of Catalonia, Spain, and composite samples were prepared for analysis. OTA was extracted, cleaned-up by immunoaffinity columns, and detected by HPLC-fluorescence detection. Mean OTA content (n = 72) was 2.17 ± 0.79 ng/g (range 1.21–4.21 ng/g, 49% occurrence). An additional sampling was done by brands (n = 45), mean OTA contamination being 2.07 ± 0.61 ng/g (range 1.30–5.24 ng/g, 95% occurrence). Coffee consumption data were obtained by means of a food frequency questionnaire. Mean coffee consumption per capita was 11.58 ± 8.73 g/person/day. OTA daily intake (DI) was estimated by means of deterministic and probabilistic methods. In both cases, estimated DI (around 0.22 ng/kg bw/day) was below the latest PTDI value of 17 ng/kg bw/day suggested by EFSA.     

Natural occurrence of aflatoxin B1, ochratoxin A and citrinin in Croatian fermented meat products

When domestic animals are exposed to mycotoxins, significant amounts of the latter shall be carried over into animal products such as milk, eggs and meat. This study was carried out in order to determine the possible presence of aflatoxin B₁ (AFB₁), ochratoxin A (OTA) and citrinin (CIT) in game sausages (n = 15), semi-dry sausages (n = 25) and fermented dry-meat products (n = 50), randomly taken from individual producers and the Croatian market. AFB₁ and OTA were quantified using ELISA, while CIT was quantified using HPLC-fluorescence detector. Out of 90 samples, the fungi most frequently isolated from dry-cured meat products were of Penicillium species, while Aspergillus was isolated from only one sample. As much as 68.88% of the samples were positive for mycotoxins. Finally, the analysis of different types of meat products resulted in OTA identification in 64.44%, CIT identification in 4.44% and AFB₁ identification in 10% of the samples. The maximum OTA concentrations established in the commercial sausage samples equalled to 7.83 μg/kg, while that of AFB₁ amounted to 3.0 μg/kg. Generally, although OTA was detected in all three types of products in different percentage shares, mutual differences were not statistically significant (P > 0.05).     

The incidence and distribution of ochratoxin A in Daqu,a Chinese traditional fermentation starter

Daqu, a traditional starter culture mainly used to produce Chinese liquor and vinegar, is spontaneously fermented by diverse bacteria, yeasts and filamentous fungi under thermophilic condition. Therefore, mycotoxins may exist in Daqu, resulting in the contamination of end-foods. Ochratoxin A (OTA), a mycotoxin produced by certain species of Aspergillus and Penicillium, is not known whether existing in Daqu. However, specific method to detect OTA as well as OTA occurrence in Daqu has not been reported so far. With this in mind, a new method was developed to detect OTA in Daqu by the combination of ultrasound-assisted solid-liquid extraction (USLE), solid phase extraction (SPE) cleanup and UPLC-MS/MS. The USLE conditions of OTA from Daqu were optimized using Plackett-Burman (PB) design coupled with Box-Behnken (BB) design. Under the optimized conditions, no matrix effects were found, and the external standard method can be used to determine OTA in Daqu. The recoveries for spiked samples were 87–106% with the relative standard deviations (RSD) < 15%. The limits of detection and quantification were respectively 0.33 and 0.41 ppb. This approach was then applied to analyze 133 Daqu samples from different geographical regions in China, including 26 low temperature-, 33 medium temperature- and 74 high temperature-type Daqu. The results showed that OTA was detected in 66 samples with a maximum concentration of 28.87 ppb in low temperature Daqu, and the OTA incidence was on increase in the order of high temperature-, medium temperature- and low temperature-type Daqu. This implied that fermentation temperature is the key factor influencing OTA occurrence in Daqu. Moreover, there may be some fungi possessing the biosynthesis ability of OTA under high temperature environment (more than 45 °C).     

Mid-infrared spectroscopy for discrimination and classification of Aspergillus spp. contamination in peanuts

In this study, Aspergillus spp., common colonists in peanut, were characterized, classified and quantified using FTIR coupled with ATR accessory. FTIR-ATR spectral data of infected peanut samples were preprocessed (mean-centering, smoothing the 1st derivative), and used for the PLS regression analysis for quantitative results. Very high R² values (96.20–99.98%) together with low error of RMSEC values (0.014–0.153 Log CFU/g of peanut) were obtained. Even, the spectrum of peanut matrix was dominant at early stages of invasion (≤2.5 Log CFU/g peanut), resulting in section separation (Nigri from Flavi) and at higher population (>4 Log CFU/g, species level separation (Aspergillus alliaceus, Aspergillus caelatus, Aspergillus flavus, Aspergillus parasiticus, and Aspergillus tamari) was observed. The accuracy of correct classification increased proportionally with fungal invasion level and 100% correct classification was reached when the cell level was Log CFU/g = 4.5–5. Samples with similar secondary metabolites (toxin producers) grouped close-by in PC score diagrams for all levels of fungal growth. Results highlight the possible implementation of FTIR-ATR model to detect infected peanuts even at early stages of invasion; besides, to prove the potential separation capability in terms of species and their secondary metabolites.     

First report: Penicillium adametzioides,a potential biocontrol agent for ochratoxin-producing fungus in grapes,resulting from natural product pre-harvest treatment

Ochratoxin A (OTA) is a secondary metabolite produced mainly by Aspergillus section Nigri (Aspergillus carbonarius is the most relevant strain in the Mediterranean region with a group 2B carcinogenic effect in humans. In vivo experiments were conducted in southern France involving applying pre-harvest Stifénia^® (elicitor), Scala^® (chemical fungicide) and two other control treatments [not contaminated by A. carbonarius (OTA-PF) and not treated and artificially contaminated by OTA-PF but not treated]. The Stifénia^® and Scala^® treatments significantly reduced the OTA juice contamination so that it was under the authorised uptake OTA limit. Stifénia^® highly affected the grape fungal ecosystem with new non-Aspergillus strains isolated from grape stems and juices. In vitro antagonistic tests were performed with Stifénia^® non-Aspergillus isolates (n = 10). Three antagonistic tests were applied using different distances (3 and 5 cm in between the two microorganisms) with two different inoculation times (at the same time and with three day intervals in between). Certain strains had a positive mycelial growth effect on A. carbonarius colonies, such as Penicillium spp. and Fusarium sp. Other strains displayed a reduction effect on OTA production of OTA-PF, such as Penicillium spp. (J2, J3). Penicillium adametzioides (S3) and Penicillium expansum (J1) (at certain stages) reduced the OTA production and mycelial growth. P. expansum was excluded as a bio-control agent because of its mycotoxin production ability. The higher challenge distance between certain strains of P. adametzioides (S3) and other Penicillium strains (as J1, J2, J3 and J4; at three and seven days) reduced the secretion of OTA by OTA-PF. This OTA production reduction could possibly prevent OTA contamination prevention in the case of epidemic favourable conditions by reducing the OTA produced in grape post-harvest products (i.e., juice). This could be accomplished by applying as the elicitor one of the tested fungi with an antagonistic effect on OTA production, such as P. adametzioides (at 10 days). Certain strains, such as P. adametzioides (S3) and J2 (P. spp.) should be further investigated to determine the details of the underlying mechanism of their OTA reduction and their ecosystem effects in cases of in vivo application.     

Development of a PCR protocol to detect ochratoxin A producing moulds in food products

Ochratoxin A (OTA) is a mycotoxin produced by several Penicillium and Aspergillus species growing in food commodities. To prevent OTA in foods it is necessary to have rapid and specific methods for early detection of producing moulds regardless of species and genera. In this work a PCR method to detect ochratoxigenic moulds has been developed. For this purpose, 75 mould strains belonging to species usually reported in food products were used. Their OTA production was checked by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A specific amplicon of 459 bp was detected by using the designed PCR protocol only in the OTA producing strains. The detection limit of the developed PCR protocol was estimated for 25 pg of mould DNA from pure cultures and from about 10²–10⁴ cfu/g when it was evaluated directly on artificially inoculated food. Its functionality in naturally infected samples was also demonstrated. In conclusion, the developed PCR method could be used for detecting ochratoxigenic moulds in foods and consequently for monitoring these moulds in the HACCP programs.     

Development of a colloidal gold strip for rapid detection of ochratoxin A with mimotope peptide

Ochratoxin A (OTA), a mycotoxin mainly produced by some Aspergillus and Penicillium species, is found in cereals, coffee, wine, pork and grapes. The kidney and liver are the target organs of OTA, resulting in teratogenicity, carcinogenicity, and mutagenicity. To avoid the risk of OTA consumption, raw materials should be identified and removed from distribution. Current procedures for detection of OTA are time-consuming and involve sophisticated equipment. Furthermore, materials containing OTA is a biohazard for manufacturers and consumers. In this study, a rapid, inexpensive, and user-friendly lateral flow strip assay ideally suited for on site testing of OTA was developed. Moreover, mimotope peptide capable of mimicking OTA by panning from a M13 phage-displayed random seven-peptide was used instead of OTA–protein conjugate. Ten ppb of OTA was detected in 10 min by this new strip. The results indicated that a rapid method without using the mycotoxin, but using mimotope peptides was developed to screen OTA; related methods also can be developed to screen other mycotoxins.     

Effect of sulfur dioxide and ethanol concentration on fungal profile and ochratoxin a production by Aspergillus carbonarius during wine making

Fungal profiles and ochratoxin A (OTA) accumulation during wine making were investigated using five different wine grape cultivars, Cabernet Sauvignon, Pinot Noir, Merlot, Syrah and Petit Verdot and the intrinsic influences caused by sulfur dioxide, ethanol and combine effect of ethanol and reducing sugar were analyzed using Cabernet Sauvignon and inoculation of Aspergillus carbonarius. Aspergillus spp. and Penicillium spp. were found as the major fungi in all winemaking processes and were highly correlated with OTA accumulation in wine. Most fungi died and OTA production decreased after 48 h of alcoholic fermentation, being consistent with the period when ethanol accumulation increased. The addition of SO₂ significantly inhibited the growth and OTA production of A. carbonarius with complete inhibition at 500 mg/L. When the ethanol concentration in the must increased to the range of 2–4%, growth and OTA production of A. carbonarius were significantly inhibited. Reducing sugar concentration had no significant effect on the growth and OTA production of A. carbonarius within the levels changing during the winemaking. Therefore, the increase of ethanol concentration played an important role in causing the decrease of fungal contamination and OTA accumulation during winemaking.     

Natural occurrence of ochratoxin A in Tunisian cereals

Ochratoxin A (OTA) is a mycotoxin produced by several fungal species from Aspergillus and Penicillium genera. It is widespread in food and feed and its occurrence has been reported in cereals, cereal-derived products, dried fruits and spices. This mycotoxin was implicated in several human and animal pathologies such as the Balkan Endemic Nephropathy (BEN) and the Tunisian Chronic Interstitial Nephropathy (CIN) of unknown cause. In Tunisia, a clear correlation has been established between the consumption of OTA contaminated food and the induction of specific pathologies. Thereby, OTA was detected in human blood and tissues. The aim of our study was to investigate the presence of OTA in widely consumed cereals commercialized in Tunisia. The analytical methods used in our study involved the extraction of OTA by acidified toluene, immunoaffinity (IAC) clean-up and HPLC quantification with fluorescence detection. Levels and percentages of OTA contamination in different types of cereals, 110 wheat, 103 barley, 113 sorghum and 96 rice samples, were evaluated with incidences of 38%, 40%, 38% and 28%, respectively. The average of contamination by OTA found were 55, 96, 44 and 117 μg/kg, respectively, for wheat, barley, rice and sorghum. Our results showed that contamination percentages and levels in the period from 2004 to 2005 were higher then usual norms (5.0 μg OTA/kg) established by the European commission in 2002. The present report is the first one ever carried out on the natural occurrence of OTA in cereals, largely consumed by the Tunisian population.     

Effect of Penicillium nordicum contamination rates on ochratoxin A accumulation in dry-cured salami

Fungal starter, such as Penicillium nalgiovense, are commonly used to inoculate sausages before seasoning process. However, Penicillium nordicum, a well-known ochratoxin A (OTA) producer frequently isolated from seasoning rooms, could colonize the casing surface during the early stage of production. The relationship between OTA accumulation and simultaneous inoculation of P. nalgiovense and P. nordicum at different rates was evaluated. After 14 days of seasoning, the persistence of P. nordicum was assessed by LAMP assay revealing its capability to colonize and grow on salami surface at all the contamination rates. At the end of seasoning, OTA was accumulated both in mycelium and dry-cured meat when P. nordicum contamination rate ranged from 25% to 100% of inoculum, while no OTA was detected in dry-cured meat at 2.5% and 0.25%. Results demonstrated that contamination of fungal starter by P. nordicum could represent a serious concern during salami production and therefore represents an important critical point to be monitored.     

Development of a Real Time PCR system for detection of ochratoxin A-producing strains of the Aspergillus niger aggregate

Members of Aspergillus section Nigri are distributed worldwide, being considered as common food spoilage fungi. Some species of this section produce ochratoxin A (OTA), mainly Aspergillus carbonarius and several members of the Aspergillus niger aggregate. Detection of ochratoxigenic A. niger aggregate strains is important to prevent OTA contamination in foodstuffs. A new Real Time PCR procedure has been developed for the rapid and specific detection and quantification of ochratoxin A-producing strains of the A. niger aggregate. Two specific primers delimiting a 120 bp fragment and a probe were designed and directed to a polyketide synthase (PKS) from A. niger CBS 513.88 genome. This PKS has a strong similarity to PKS of A. ochraceus fragment involved in ochratoxin biosynthesis. Specificity was confirmed by testing primers towards purified DNA from 91 fungal strains, including reference and food isolates. The SYBR-Green and the TaqMan approaches developed allowed the specific detection only of ochratoxigenic strains of the A. niger aggregate. All other analyzed food related fungi gave negative results. This is the first report on a Real Time PCR system for the detection of OTA-producing strains of the A. niger aggregate.     

Evaluating the combined effect of water activity,pH and temperature on ochratoxin A production by Aspergillus ochraceus and Aspergillus carbonarius οn culture medium and Corinth raisins

The objectives of this study were: (a) to evaluate the potential of the ELISA method in the determination of the produced OTA by Aspergillus ochraceus and Aspergillus carbonarius in malt extract agar (MEA) at different pH (3.9, 5.1, 5.9, 6.8), water activity (a_w) (0.87, 0.93, 0.99), and temperature (10, 15, 20, 25, 30, 40 °C) levels, providing a rapid screening for the optimum and marginal conditions of OTA production, (b) to comparatively evaluate the performance of ELISA and HPLC method, and (c) to evaluate the ability of A. ochraceus to produce OTA in rehydrated Corinth raisins during storage for 36 days. Two independent experiments were carried out to estimate OTA production on MEA and Corinth raisins. The produced OTA was evaluated qualitatively by the ELISA method and selected cases were verified by HPLC. The levels of OTA decreased with water activity, whereas pH seemed to have no specific effect. Furthermore, A. ochraceus produced maximum amounts of OTA on raisins at the 24th day of incubation, indicating that the endogenous microflora may restrictively affect OTA production. The knowledge of optimal and marginal levels of ecological factors in order to optimise post-harvest and storage of food products may significantly affect the production of OTA. Moreover, endogenous microflora of certain foodstuffs may cause OTA detoxification and consequently reduction of OTA levels; a fact that has to be taken into account in food commodities such as raisins, grapes, and wine.