In vivo stability of the complex ochratoxin A – Saccharomyces cerevisiae starter strains

In recent years, several studies have focused on the role of yeasts as adsorbing tools to remove ochratoxin A (OTA) in musts and wines; however, no data are available on the stability of OTA-yeast complex. The present study was designed to assess the ability of five genetically distinct Saccharomyces cerevisiae strains (W13, W28, W46, W47, and Y28), previously studied for their oenological traits and in vitro OTA removal ability, to stably bind the toxin in Uva di Troia grape must (a red variety from Southern Italy). In addition, the effect of temperature, sugar concentration, and addition of ammonium salts on OTA binding was investigated.The highest binding was observed for S. cerevisiae W46 and W28 (42.79–76.44 and 34.51–70.17%, respectively); the other strains (W13, W47 and Y28) removed OTA by 20.34–53.79%. The binding was reversible and the extent of process was strongly affected by the kind of strain. Although the strains W46 and W28 removed OTA efficiently, they did not irreversibly bind OTA (ca. 80–85% release). On the other hand, the complex “S. cerevisiae W13-OTA” exhibited the greatest stability (ca. 55% release).The results obtained may be relevant not only to improve wine safety by integrating the binding ability in the criteria of selection of wine starters, but also for a more general biotechnological purpose, because of the increasing interest in the bioremediation of musts and wines using yeasts.     

Aflatoxins and ochratoxin A in Brazilian paprika

Seventy paprika samples collected in the city of São Paulo, Brazil, from January to April 2006 were analysed for aflatoxins and ochratoxin A (OTA) using an immunoaffinity column clean-up and HPLC-FLD. For aflatoxins, the limit of quantification (LOQ) were 0.23, 0.23, 0.45 and 0.45 μg/kg for AFB₁, AFB₂, AFG₁ and AFG₂, respectively. For OTA the LOQ was 0.80 μg/kg. Aflatoxins were found in 82.9% of samples and AFB₁ was detected in 61.4% at levels ranging from 0.5 to 7.3 μg/kg with mean concentration of 3.4 μg/kg. OTA was found in 85.7% at levels ranging from 0.24 to 97.2 μg/kg with mean concentration of 7.0 μg/kg.     

Fate of ochratoxin A during wheat milling and some Chinese breakfast processing

To estimate the dietary intake of ochratoxin A (OTA), information on the fate during wheat processing is needed. The wheat contaminated with OTA at both levels (93.22 and 248.93 μg/kg) was artificially inoculated by Aspergillus ochraceus. Concentrations of OTA were 1.43–1.66 times higher in bran than whole wheat and 1.27–1.50 times higher in shorts than bran. A concentration reduction of 43.29–55.16% for OTA in flour was observed, with the final concentrations of 41.80 and 138.33 μg/kg in flour respectively. Whereas, OTA was not transferred or decomposed during milling, as the total amount of OTA in all milling fractions didn't decreased. The further losses of OTA were various depending on different processes. The concentration of OTA increased by 11–16% when the flour was processed into Chinese steamed bread (CBS), with the OTA concentrations of 49.2 and 161.35 μg/kg in CBS. It revealed fermentation and steaming increased the OTA levels in CBS. The loss of OTA was small in the Chinese fried bread sticks (CFBS) (38.84 and 129.02 μg/kg), being of 6.73–9.63%. A higher reduction of 23.27–23.92% for OTA was detected in the noodles processing, with the final concentration of 32.98 and 108.24 μg/kg in noodles respectively. The result revealed that the temperature, pH values and strains involved the processes exerted different effects. High temperature (180 °C) and alkaline condition (pH > 7) facilitated to the removal of OTA, while fermentation (with dry yeast) increased the OTA levels in products.     

Study of Saccharomyces cerevisiae W13 as a functional starter for the removal of ochratoxin A

In this paper, a wild strain of Saccharomyces cerevisiae (W13) was studied for its OTA-removal ability, growth pattern and alcoholic fermentation profile at two different temperatures (25 and 30 °C) and two different sugar levels (200 and 250 g l⁻¹), with or without supplementation of medium with diammonium phosphate (DAP). A commercial strain (Lallemand EC1118) and a collection isolate (DBVPG 6500) of S. cerevisiae were also studied. All the strains were able to conclude fermentation, with S. cerevisiae W13 producing higher ethanol and glycerol contents; moreover, the wild strain was able to remove OTA (reduction of 6–57.21%), with the highest removing effect observed at 30 °C with 250 g l⁻¹ sugar and after 3 days.In addition, S. cerevisiae W13 was studied for its technological and qualitative traits, as it showed a high tolerance to single and combined stress conditions, β-d-glucosidase, pectolytic and xylanase activities, a low level of hydrogen sulphide production, a low-to-medium parietal interaction with phenolic compounds and no biogenic amines formation.The findings of this work have an applicative value for the potentiality of using S. cerevisiae W13 as functional starter for the production of wines with improved qualitative and food safety characteristics.     

Aflatoxins and ochratoxin A in dried chestnuts and chestnut flour produced in Italy

The occurrence of aflatoxins and ochratoxin A in chestnut products made in Italy was surveyed. Thirty-seven samples of chestnut flour and fourteen of dried chestnuts were collected from retail outlets located in northern Italy. After extraction and purification through an immunoaffinity column, aflatoxins and ochratoxin A were analysed using both HPLC-FLD and HPLC-MS/MS. The mycotoxin contamination found in this survey was widespread and remarkable. As regards aflatoxins, the incidence of aflatoxin B₁ was 62.2 and 21.4% in chestnut flour and dried chestnuts, respectively; in the same products, the percentage of samples exceeding the value of 2.0 μg kg⁻¹ for aflatoxin B₁ (maximum limit fixed by EC Regulation 165/2010 in dried fruits) was 24.3% and 7.1%. A widespread and high incidence of AFG₁ was also found (40.5%). The maximum values for aflatoxin B₁ and total aflatoxins were 67.88 and 188.78 μg kg⁻¹, respectively (chestnut flour sample). Ochratoxin A occurred in all samples, showing very high values (mean 12.38 and 13.63 μg kg⁻¹ for chestnut flour and dried chestnuts, respectively); the percentages of samples exceeding the limit of 10 μg kg⁻¹ (EU limits for dried vine fruit) were 64.9 and 42.8% for chestnut flour and dried chestnuts, respectively. The maximum value was 65.84 μg kg⁻¹ (dried chestnut sample).     

Incidence of aflatoxins, ochratoxin A and zearalenone in tunisian foods

A total of 209 samples of different groups of foods widely consumed by the Tunisian population were collected during 2004–2005 years. Samples were analyzed for contamination with aflatoxins, ochratoxin A and zearalenone, using competitive enzyme-linked immunosorbent assay (ELISA). The predominant mycotoxin was ochratoxin A with a mean level of 3.5 ± 5.3 ng g⁻¹ in 59.8% of studied samples. Furthermore, Aflatoxins were detected in all analyzed commodities with a contamination frequency of 50.5%. In addition, aflatoxin B1 was found in 37% of the samples. The zearalenone was detected around 15% with a mean level of 10.4 ± 11.8 ng g⁻¹. Species, dried fruits and sorghum were the most contaminated samples by aflatoxin and ochratoxin mycotoxins, whereas Rice was the least contaminated commodity. The most frequent mycotoxins co-occurrence included aflatoxins and ochratoxin A, which have been detected in 33.8% of analyzed samples. Furthermore, the simultaneous contamination by aflatoxins, ochratoxin A and zearalenone was observed in 7.2% of studied samples.     

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.     

Investigation of ochratoxin A in Syrian consumed baby foods

An investigation on the occurrence of ochratoxin A (OTA) in baby food products consumed in Syria was conducted. A total of 42 samples of baby food formulae, including 30 cereal-based baby food and 12 fruit-based baby food formulae were analyzed using immunoaffinity columns (IAC) for sample clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD). According to the results in the present study, the mean recoveries of OTA from spiked cereal-based baby food and fruit-based baby food samples were in the range of 92.85–107.4% and 83.3–87.86%, respectively. While the limit of detection (LOD) and limit of quantification (LOQ) for OTA were 0.038 and 0.11 µg/kg for cereal-based baby foods and 0.05 and 0.17 µg/kg for fruit-based baby foods, respectively. Analytical results showed that 40.48% of the analyzed samples were found contaminated with OTA. Among the OTA contaminated samples, 43.33% samples of cereal-based baby food, and 33.33% samples of fruit-based baby food contained mean values of 0.094 and 0.093 µg/kg of OTA, respectively. Our findings show that contamination levels of OTA in all samples were lower than the permitted level set by European Commission Regulation.     

Aflatoxin M1 and ochratoxin A in human milk in Ribeirão Preto-SP,Brazil

The objective of this study was to determine the extent of aflatoxin M₁ (AFM₁) and ochratoxin A (OTA) contamination in human breast milk in the city of Ribeirão Preto, São Paulo State, Brazil. During 2012, 100 samples of human milk were collected at the local Human Milk Bank. The method comprised, immunoaffinity column purification and isolation, liquid chromatography separation and fluorescence detection. The average percentage recoveries of AFM₁ and OTA spiked at 20 and 50 ng/L in control human milk were 78.1 ± 11.7% and 73.7 ± 9.6%, respectively. The average relative standard deviations of AFM₁ and OTA spiked at the same levels were 11.7 and 9.6% respectively. The limits of detection was 0.3 ng/L for AFM₁ and OTA. The limit of determination was 0.8 ng/L for both mycotoxins. This method was used to analyze 100 human milk samples, of which, two samples were found to contain AFM₁ at level greater than 0.3 ng/L. OTA was detected in 66 samples (66%), wherein 32 were above the limit of detection and 34 were in the range of from 0.8 to 21 ng/L. Results of our study indicate that breast-fed Brazilian infants had only an insignificant exposure to AFM₁ and OTA.     

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.     

Inhibition of Aspergillus carbonarius and fungal contamination in table grapes using Bacillus subtilis

Aspergillus carbonarius is a major producer of ochratoxin A in grapes, causing fungal decay and posing a significant threat to human health. Bacillus subtilis CCTCC M 207209 was used to inhibit the growth of A. carbonarius CCTCC AF 2011004, an ochratoxin A-producing strain previously isolated from grapes. The inhibition effect in vitro was tested in potato dextrose agar medium (PDA), while in vivo effects were examined in grape berries of three different grape cultivars: Thompson Seedless, Kyoho, and Red Earth. Inhibitory effects were evaluated according to colony size in PDA and rotten spots in grape berries when A. carbonarius and B. subtilis were inoculated simultaneously and cultivated at 0 °C, 25 °C, or 30 °C. B. subtilis liquid cultures with and without cells and volatile products were used in the analyses. Significant inhibition of A. carbonarius was observed in all samples treated with B. subtilis liquid cultures, especially those subjected to cell-free culture treatment. No inhibition was observed for A. carbonarius treated with the volatile products of B. subtilis. The inhibition was the most significant in Red Earth grapes, followed by the Kyoho then Thompson Seedless varieties, when the same fraction of B. subtilis culture was used. Significant inhibition was also observed for other fungal contaminants in grapes when B. subtilis liquid culture supernatant was used. This study reveals the potential of B. subtilis for inhibiting contamination of OTA-producing A. carbonarius and other fungi in table grapes.     

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).     

Occurrence of toxigenic fungi and ochratoxin A in Ethiopian coffee for local consumption

Information on the presence of ochratoxin A (OTA) in local Ethiopian coffee is scarce. Therefore, the objective of this study was to type the fungal and mycotoxin contamination levels in Ethiopian coffee consumed by the local community. Coffee samples were collected from six major coffee growing districts in Jimma zone of Oromiya regional state in Ethiopia. Total fungal incidence mounted up to 87% and Aspergillus (79%), Fusarium (8%) and Penicillium (5%) were the predominant toxigenic genera. OTA producing species A. westerdijkiae, Aspergillus ochraceus, and Aspergillus steynii were identified for the first time in Ethiopia and were shown to be the predominant species while aflatoxin (AF) producing Aspergillus species were scarce. Using an in vitro approach, the OTA production potential was assessed under standardized conditions. Based on this experiment, it was concluded that Aspergillus westerdijkiae isolates were clearly the most potent producers of OTA. The median OTA level in the locally sold Ethiopian coffee was 1.53 μg/kg. Notwithstanding this low median value, significant differences in fungal and toxin incidences were observed between the different coffee processing types, coffee sample types, and storage characteristics.     

Survey of aflatoxin B1 and ochratoxin A occurrence in traditional meat products coming from Croatian households and markets

The aim of this study was to investigate the occurrence of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in different traditional meat products circulating on Croatian markets, produced by a large number of households situated in different Croatian regions. The study involved a total of 410 samples of traditional pork meat products in terms of hams (n = 105), dry fermented sausages (n = 208), bacon (n = 62) and cooked sausages (n = 35), collected over four years period (2011–2014). Mycotoxin concentrations were quantified and confirmed using validated immunoassay method (ELISA) and high performance liquid chromatography with fluorescence detection (HPLC-FLD), respectively. The maximal observed OTA level in the fermented sausages and hams was around 5 times (5.10 μg/kg) to 10 times (9.95 μg/kg) higher than the maximal recommended level (1 μg/kg) stipulated for pork products in some EU countries. AFB₁ levels found in any given meat product analysed within this frame were not significantly higher (p > 0.05) than the applied method limit of detection. The results showed an occasional mycotoxin contamination of traditional meat products, especially that by OTA, pointing that to avoid such contamination meat and meat products on households should be produced and processed under standardised and well-controlled conditions.     

Co-occurrence of aflatoxins and ochratoxin A in spices commercialized in Italy

A total of 130 spice samples coming from India, China, South America, USA, Northern Africa, Europe and Sub-Saharan Africa were collected in different stores of Northern Italy. They were analysed for aflatoxins (AFs: AFB₁, AFB₂, AFG₁, AFG₂) and ochratoxin A (OTA) content by liquid chromatography with mass spectroscopy and positive electrospray ionization (LC/ESI-MS/MS), and HPLC with fluorescence detector (FLD), respectively. The analysis showed that 20 (15.4%) and 31 (23.8%) out of 130 samples were contaminated with AFs and OTA, respectively. A low level of total AFs contamination was found in the positive samples, the average concentration was 0.64 ng g⁻¹, far below the maximum threshold admitted by the European legislation (5 ng g⁻¹ for AFB₁, and 10 ng g⁻¹ for total aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂). A higher incidence of OTA was found in chili (60.0%) more than in pepper (13.3%), ranging from 2.16 to 16.35 ng g⁻¹, and from 1.61 to 15.85 ng g⁻¹, respectively. Moreover, three spice samples (2.3%) contaminated by OTA trespassed the threshold admitted by the European Regulation (EC, 2010). The co-occurrence of OTA and AFs in spices was detected in 6 out of 130 samples (4.6%), ranging from 1.61 to 15.85 ng g⁻¹ and from 0.57 to 3.19 ng g⁻¹, respectively.     

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.     

A simple and rapid biosensor for ochratoxin A based on a structure-switching signaling aptamer

A fast and reliable sensing platform has been developed for the detection of mycotoxin ochratoxin A (OTA) based on a target-induced structure-switching signaling aptamer. In the absence of target, a fluorescein-labeled OTA aptamer hybridizes to a complementary DNA strand containing a quencher moiety, bringing the fluorophore and the quencher into close proximity for highly efficient fluorescence quenching. Upon OTA addition, a conformational change in the aptamer releases the quencher-containing DNA strand, generating a strong concentration-dependent fluorescent signal. Using this technique, the entire detection and analysis process of OTA can be completed within 1 min. Under optimized assay conditions, a wide linear detection range (from 1 to 100 ng/mL) was achieved with a detection limit down to 0.8 ng/mL. Additionally, the proposed assay system exhibited high selectivity for OTA against other mycotoxins (aflatoxin B₁ and zearalenone) and limited interference from the structural analog ochratoxin B. The biosensor was also applied to a non-contaminated corn material spiked with a dilution series of OTA, obtaining recoveries from 83% to 106%. Utilization of the proposed biosensor for quantitative determination of mycotoxins in food samples may provide significant improvements in quality control of food safety through a simple, rapid, and sensitive testing system for agricultural products monitoring.     

Identification of ochratoxin A in Chinese spices using HPLC fluorescent detectors with immunoaffinity column cleanup

Spices can be heavily contaminated with Penicillium verrucosum and Aspergillus ochraceus, which develop during cultivation and after harvest if relative humidity is not controlled during storage. The present study was undertaken with the aim to provide an analytical method to detect OTA in spices commercialized in China, specifically to develop a simple and rapid method for the determination of Ochratoxin A content in spices via immunoaffinity column and high performance liquid chromatography with fluorescence detection (HPLC-FLD). The recovery (75.0–102.0%) and repeatability (RSD < 12) are acceptable according to the requirements of the Commission Regulation (EC) No. 401/2006. The method is accurate, sensitive and rapid. A total of 480 spice products were analyzed with the amended analytical methods used in our study, which involved the extraction of OTA, immunoaffinity cleanup and high performance liquid chromatography determination with fluorescence detection. This report is the first describing the OTA presence in retail spices (pepper, chili, prickly ash, cinnamon, aniseed, fennel, curry powder and cumin) in China. The percentage of OTA contamination in spice product is 9.6%, and two chili products were above the levels permitted by the EU for safe consumption. Surveillance of a large number of suspected spice products should be continuous and widespread.     

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.     

Survey of aflatoxins and ochratoxin A in retail market chilies and chili sauce samples

The aim of this study was to determine the levels of the aflatoxins (AFs) and ochratoxin A (OTA) in 312 samples of whole chili, chili powder, crushed chili and chili sauce samples from suburbs, open market and food restaurants of Punjab, Pakistan. The analysis was carried out using HPLC with fluorescence detector, after immunoaffinity column clean-up. The results have shown that 176 out of 312 (56.4%) samples were to be positive with AFs and 126 out of 312 (40.4%) sample of chilies were found to be contaminated with OTA. Total mean level of AFB₁ and total AFs in chilies were 12.50 ± 1.91, 15.16 ± 2.22 μg/kg, respectively. The total mean level of OTA in chilies was found 16.68 ± 2.58 μg/kg, ranged from LOD to 120.9 μg/kg. Sample 39.5, 26.3 and 32.7% of chilies were found containing level of AFB₁, total AFs and OTA, higher than the recommended limits for EU, respectively. The dietary levels of 3.26, 3.52, and 3.84 μg/kg were determined for AFB₁, total AFs and OTA in chilies. The incidence and levels of AFs and OTA in chilies are higher and could pose serious health hazards for consumers.