Mycobiota and co-occurrence of mycotoxins in Capsicum powder

This study aimed to: (1) determine the mycobiota of Capsicum powder samples, paying a special attention to the mycotoxigenic moulds; (2) evaluate the contamination levels of aflatoxins (AF), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), T2 and HT2 toxins in those samples. Thirty-two samples were obtained through the methods of sampling established by the European Union legislation. Aspergillus and Eurotium were the most frequently found genera. Aspergillus section Nigri had the higher relative frequency in the samples, A. niger aggregate being the most representative group of this section. Other potentially mycotoxigenic Aspergillus, Fusarium and Penicillium species were found, but in a lower frequency.Co-occurrence of mycotoxins was confirmed in the 32 Capsicum powder samples. All samples were contaminated with AF and OTA, 27% with ZEA (36% of chilli and 18% of paprika samples), 9% with DON (18% of chilli and 6% of paprika samples), 6% with T2 (18% of chilli samples) and none of the samples contained HT2.Although in the present study the most common genera found (Aspergillus and Eurotium) belong to storage moulds, some field fungi such as Fusarium spp. were also found, and their toxins were sometimes detected. This fact supports the hypothesis that mycotoxin contamination of Capsicum products may occur both in the field and/or during storage.     

Biodiversity of Aspergillus section Nigri populations in Argentinian vineyards and ochratoxin A contamination

Aspergillus section Nigri are described as the main source of ochratoxin A (OTA) contamination in grapes and wine worldwide. The knowledge of the factors affecting grape contamination by species included in this section and OTA production is essential to be able to reduce their presence, not only to improve wine quality, but also to maintain their safety. Therefore, the aims of this study were to determine the incidence of Aspergillus section Nigri species harvested in different grape-growing regions from Argentina, their ability to produce OTA, to correlate with meteorological conditions and geographical coordinates with their prevalence and to evaluate the OTA natural occurrence in grapes and wines. The morphological identification showed that Aspergillus niger aggregate species were the most prevalent ones, followed by Aspergillus carbonarius and Aspergillus uniseriate. These populations were confirmed through using AFLP markers and sequencing and, Aspergillus tubingensis was separated from A. niger aggregate. Climatic factors, altitude, longitude and latitude have influenced on the distribution of species included in the section. A. carbonarius and A. niger were OTA producers but differed in their OTA producing ability. Temperature was the factor which influenced the most over the highest incidence of A. carbonarius in La Rioja and San Juan regions. The trellis system in vineyards and drip irrigation also influenced the species isolation. The OTA levels detected in grapes and wines were low, but grape variety was more important in susceptibility to fungal infection and OTA levels.     

Mycoflora and natural occurrence of aflatoxin,cyclopiazonic acid,fumonisin and ochratoxin A in dried figs

Mycoflora, the mycotoxigenic properties of moulds, and natural contamination with mycotoxins such as aflatoxins (AFs), cyclopiazonic acid (CPA), fumonisin B₁ (FB₁) and ochratoxin A (OTA) were investigated in dried figs. Dry fig samples were collected from orchards during the drying stage in the Aegean Region of Turkey. Fungal isolates were identified using morphological, chemical as well as molecular methods. Mycotoxigenic characteristics of moulds were assessed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Mycotoxins except CPA (by TLC) were determined by HPLC. All the fig samples were contaminated with moulds and 94.7% contained one or more mycotoxigenic species. The most prevalent moulds present in dried figs belong to the Aspergillus section Nigri members, being 93.9% positive for the samples, followed by Fusarium spp., Aspergillus section Flavi and Penicillium spp. On the other hand, Fusarium spp. had the highest count and the number of fumonisin producing Fusarium was also high. A total of 48% of 115 dried fig samples contained OTA (range?=?0.1–15.3?ng?g^?1), 74.7% of the samples had FB₁ (range?=?0.05–3.65?mg?kg^?1), 10.0% of the samples had aflatoxin (range?=?0.1–763.2?ng?g^?1) and 24.3% of the samples were tentatively identified as being contaminated with CPA (range?=?25–187?ng?g^?1). Dried fig samples were contaminated with one (33.0%), two (47.0%), three (5.2%) and four mycotoxins (3.5%). A total of 11.3% of dried fig samples were not contaminated with any of the four mycotoxins. To the best of our knowledge, CPA and fumonisin have been found for the first time in dried figs.     

Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.     

A survey on distribution and toxigenicity of Aspergillus section Flavi in poultry feeds

Thirty-five samples of poultry feeds and corresponding raw materials (maize, soybean and meat meal) from a processing plant were analyzed to evaluate the distribution and toxigenicity of Aspergillus section Flavi isolates. Mycological analysis of the samples indicated the presence of five fungal genera (Aspergillus, Penicillium, Fusarium, Cladosporium, and Eurotium). Aspergillus flavus was the predominant species being present in 48.5% of the analyzed samples. Ninety-one isolates belonging to Aspergillus section Flavi were isolated; ninety were identified as A. flavus and only one as A. parasiticus. Fifty-seven isolates were capable of producing sclerotia, 41 were identified as L-type strains and 16 as type S. Fifty-seven percent of the isolates produced AFB₁ levels ranging from 0.05 μg/kg to 27.7 μg/kg whereas 86.8% produced CPA from 1.5 μg/kg to 137.8 μg/kg. L-strains produced from 0.05 to 14.8 μg/kg of aflatoxin and type S produced levels from 0.05 to 1.65 μg/kg. No significant differences in CPA production among S- and L-strains were observed. Sclerotial isolates produced AFB₁ levels ranging between 0.05 and 27.7 μg/kg and CPA levels from 3.8 to 47.3 μg/kg. More than half of the A. flavus isolates were able to produce AFB and CPA simultaneously. Twenty percent of the 35 samples were contaminated with aflatoxin B₁ whereas 34.3% were contaminated with CPA. The high rate of CPA producing isolates represents a potential risk of contamination with this toxin in poultry feeds.     

Capsaicin,an inhibitor of Ochratoxin A production by Aspergillus section Nigri strains in grapes (Vitis vinifera L.)

ABSTRACT

Food decay by spoilage fungi leads to significant economic losses and hazards to consumers’ health due to the potential of mycotoxin occurrence. Ochratoxin A (OTA) is a mycotoxin known as nephrotoxic and carcinogenic to humans. Natural capsaicin was evaluated for its effectiveness against the growth of five Aspergillus section Nigri strains and accumulation of OTA in inoculated black grapes. Results showed that capsaicin was effective in inhibiting fungal growth and OTA production by new four Aspergillus section Nigri strains (ATHUM 6997, 6998, 6999, 7000) and by Aspergillus carbonarius as well. Moreover, capsaicin addition exhibited maximum inhibition of OTA produced by ATHUM 6997, 6998, 6999, and 7000 in black grapes at 28.9%, 8.6%, 68.4%, and 78.1%, respectively. Inhibition percentage of OTA production by A. carbonarius in grapes treated with capsaicin was estimated at 61.5%. These results suggest that capsaicin influences the OTA biosynthesis pathway of all Aspergillus section Nigri strains and therefore could be used as an effective natural preservative against OTA contamination of vineyards. Risk assessment revealed that when grapes are treated with capsaicin, consumers are less exposed to OTA.     

Occurrence of ochratoxin A and toxigenic potential of fungal isolates from Spanish grapes

This paper reports the results from an extensive survey of filamentous fungi isolated from wine‐producing grapes and their capacity to produce ochratoxin A (OTA) on Czapek Yeast Autolysate agar (CYA), in order to assess their potential for producing this toxin in grapes. Grapes were sampled from four Spanish wine‐producing regions during 2001. The fungal infection in berries increased with time, reaching 100% at harvest. A total of 386 isolates of Aspergillus section Nigri and 10 of Aspergillus section Circumdati were isolated and tested for their ability to produce OTA in CYA. 21 strains produced OTA (18 Aspergillus section Nigri and 3 Aspergillus section Circumdati). Aspergillus section Circumdati isolates produced higher amounts of OTA than Aspergillus section Nigri ones, with means of 10.76 µg g^?1 CYA and 1.42 µg g^?1 CYA, respectively. Despite this, black aspergilli are believed to be highly responsible for the OTA levels found in musts and wines, as it is more widespread in grapes. Musts (n = 40) produced from the grapes collected were analysed. 15% were found to contain OTA, concentrations ranging from 0.091 to 0.813 ng ml^?1 (detection limit: 0.07 ng ml^?1), but no correlation was found with the ochratoxigenic moulds isolated from grapes. Copyright © 2004 Society of Chemical Industry     

Ochratoxin A and the occurrence of ochratoxin A‐producing black aspergilli in stored peanut seeds from Córdoba,Argentina

Since there is no available information about the natural occurrence of ochratoxin‐producing fungi and ochratoxin A (OTA) in peanut seeds in Argentina, the aims of this work were to isolate and identify Aspergillus section Nigri and to evaluate the natural occurrence of OTA in stored peanut seeds. Likewise, the capacity to produce OTA by Aspergillus section Nigri was studied. Forty‐seven samples of peanut seeds were obtained from a storage plant in the south of Córdoba Province, Argentina. Each sample of 100 seeds was surface‐disinfected and plated onto a dichloran 18% glycerol agar (DG18). OTA was detected by HPLC. The production of OTA in strains belonging to section Nigri was tested in YES medium (2% yeast extract, 15% sucrose). Aspergillus spp., the most frequent mould, occurred in 100% of samples, followed by Penicillium spp. (87%) and Eurotium spp. (6.4%). A. flavus was isolated in 100% of the samples, followed by A. niger var. niger, A. niger var. awamori and A. carbonarius at a lower frequency, 89%, 68% and 4%, respectively. OTA was found in 32% of the peanut seed samples, with mean levels ranging from 0.5 to 170 ng g^?1. The mean recovery of the method used was 85 ± 2%. Forty‐three isolates (27%) of Aspergillus section Nigri, were found to be OTA producing strains. The highest percentage of ochratoxigenic strains (57%) was found within the A. carbonarius ssp. These results indicate a human exposure to OTA in Argentina through the ingestion of peanut seeds and peanut products. Copyright © 2006 Society of Chemical Industry     

Polyphasic characterization of Aspergillus section Flavi isolated from animal feeds in Algeria

In Algeria, little information is available on the population structure of Aspergillus section Flavi in raw materials and resultant animal feeds. A total of 172 isolates belonging to Aspergillus section Flavi were recovered from 57 animal feeds and identified on the basis of macro and micro-morphological characters, mycotoxin production and genetic relatedness. For the molecular analysis, sequencing of the calmodulin gene (CaM) and the internal transcribed spacer (ITS) regions were performed for representative isolates. Four distinct morphotypes were distinguished: Aspergillus flavus (78.5%), Aspergillus tamarii (19.2%), Aspergillus parasiticus (1.7%), and Aspergillus alliaceus (0.6%). All A. flavus isolates were of the L type and no correlation between sclerotia production and aflatoxigenicity was observed. Our results showed that 68% of the A. flavus strains produced aflatoxins B (AFB), and 72.7% were cyclopiazonic acid (CPA) producers. The three isolates of A. parasiticus were able to produce AFB and aflatoxins G but not CPA whereas, all the strains of A. tamarii produced only CPA. The obtained results revealed the presence of different species of Aspergillus section Flavi, among which were aflatoxin producers. This study provides evidence useful for considerations in aflatoxin control strategies.     

Aflatoxins and ochratoxin A and their Aspergillus causal species in Tunisian cereals

ABSTRACT

Occurrence of aflatoxins (AFs) AFB1, AFB2, AFG1, AFG2 and ochra toxin A (OTA) was studied in 65 samples of stored and freshly harvested wheat, barley and maize collected in Tunisia. The mycotoxins were simultaneously extracted and quantified by high performance liquid chromatography. Determination of AF-producing (section Flavi) and OTA-producing Aspergillus species (sections Nigri and Circumdati) was conducted in these samples by species-specific polymerase chain reaction (PCR). Results showed that most of maize samples were contaminated with AFs, data after storage showing lower values than those collected at harvest. All contaminated maize samples contained AFG1 and AFG2, among which 27.78% also had AFB1 and AFB2. This AFs pattern was consistent with the A. parasiticus toxin profile. A. flavus however showed the highest frequency in maize but was also found in barley and wheat where no AFs were detected. In contrast, OTA was neither found in maize nor in barley and only one wheat sample contained OTA. A. niger was the only OTA-producing species detected.     

Predominant mycobiota and aflatoxin content in Brazil nuts

The Brazil nut (Bertholletia excelsa) is an economically important product to the Brazilian Amazon. Currently, its marketing is being affected by the high incidence of aflatoxins (AF) produced by potentially aflatoxigenic fungi associated with its seeds. In this context, the purpose of this study was to determine which part of the nut contributes to contamination by aflatoxins and to identify the mycobiota in Brazil nut samples. Unshelled and shelled nuts were analyzed by measuring the total count of filamentous fungi (Aspergillus sections Flavi, Nigri and Circumdati) in sanitised and non-sanitised treatments. The isolates identified as Aspergillus section Flavi, the major producers of AF, were plated for determination of their aflatoxigenic potential. To perform the AF analysis, samples of Brazil nuts were treated separately. The AF from the shell and kernel were extracted by chloroform and analysed by the HPLC-FD system in isocratic mode. The Aspergillus section Flavi count was 21.67% lower. The production of AF by the isolated fungi was 30% for sanitised and 23.8% for non-sanitised samples. The concentrations obtained of AFB₁ and AFG₁ were higher than those of AFB₂ and AFG₂. The AFB₁ concentrations of shelled nuts and shell samples were 35.0 and 1.78???g/kg, respectively. AFB₂ and AFG₂ were detected only in shelled nut samples. The HPLC-FD analysis presented limits of detection (LOD) and quantification (LOQ) of 0.2 and 0.4???g/kg, respectively.     

Evaluation of food grade antioxidant formulation for sustained antifungal,antiaflatoxigenic and insecticidal activities on peanut conditioned at different water activities

The aim of this study was to investigate antifungal and insecticidal activity of two microencapsulated antioxidants: 2(3)-tert-butyl-4 hydroxyanisole (BHA) and 2,6-di(tert-butyl)-p-cresol (BHT) against Aspergillus section Flavi and Oryzaephilus surinamensis (L.), a vector carrier of aflatoxigenic fungi on stored peanuts. Susceptibility of Aspergillus section Flavi, insects, and aflatoxin B₁ accumulation in sterile peanut kernels conditioned at two different water activities (a_w) (0.83 a_w and 0.95 a_w) was determined with different doses of antioxidant formulations (10, 20 and 30 mM) during 45 days. Moreover, Aspergillus section Flavi isolation frequency from live and dead insects was evaluated. The BHA formulation completely inhibited Aspergillus section Flavi development regardless of a_w and doses assayed. Antifungal effect of microencapsulated BHT was highly dependent on a_w, with 86–100% fungal inhibition at 20 and 30 mM, at the lowest a_w (0.83 a_w) and at the end of the experiment. No aflatoxin accumulation was detected in samples treated with the BHA formulation. In general, low levels of Aspergillus section Flavi were detected in dead insects. Our results show efficacy for 45 days, in addition microencapsulated BHT could be an alternative to control peanut pests in dry kernels.     

Phylogenetic characterization and ochratoxin A--fumonisin profile of black Aspergillus isolated from grapes in Argentina

Aspergillus section Nigri populations isolated from seven growing regions from Argentina were characterized by sequencing in order to identify species responsible for production of ochratoxin A (OTA) and fumonisins (FB_s). Sequences of genes encoding calmodulin, β-tubulin, the second largest subunit of RNA polymerase II and translation elongation factor 1 alpha were analysed. The phylogenetic analysis showed the presence of six lineages: A. carbonarius, A. tubingensis, A. niger, A. japonicus, A. homomorphus and A. foetidus grouped in four major clusters. The molecular tools used allowed the identification for the first time of A. homomorphus from vineyards. OTA production confirmed the importance of A. carbonarius as the main ochratoxigenic species isolated and, to a variable degree, of A. niger and A. tubingensis, which were by far the most commonly occurring species on grapes in Argentina. The only strains able to produce OTA and fumonisins (B₂-B₄) belong to the A. niger cluster.     

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.     

Incidence of lactic acid bacteria and Aspergillus flavus in brewer's grains and evaluation of potential antifungal activity of these bacteria

Feed destined for animal production as brewer's grains can be contaminated by Aspergillus section Flavi species. Lactic acid bacteria (LAB) play a defining role in the preservation and microbial safety of fermented foods. The objective of this study was to study the incidence of lactic acid bacteria, Aspergillus section Flavi and AFB₁ in brewer's grains and the preliminary antifungal activity of native LAB in vitro. LAB and aflatoxigenic Aspergillus were found in high counts in brewer's grains used as raw material for pig feedstuff. However, AFB₁ had low AFB₁ natural incidence in samples. In vitro antifungal activity of LAB isolated showed that all bacteria tested inhibited two Aspergillus flavus strains assayed. The high incidence of LAB could be inhibiting the AFB₁ production in by-products obtained from the beer industry. LAB strains with excellent antimicrobial activity were also found in this substrate.     

Predominant mycobiota and aflatoxin content in Brazil nuts

The Brazil nut (Bertholletia excelsa) is an economically important product to the Brazilian Amazon. Currently, its marketing is being affected by the high incidence of aflatoxins (AF) produced by potentially aflatoxigenic fungi associated with its seeds. In this context, the purpose of this study was to determine which part of the nut contributes to contamination by aflatoxins and to identify the mycobiota in Brazil nut samples. Unshelled and shelled nuts were analyzed by measuring the total count of filamentous fungi (Aspergillus sections Flavi, Nigri and Circumdati) in sanitised and non-sanitised treatments. The isolates identified as Aspergillus section Flavi, the major producers of AF, were plated for determination of their aflatoxigenic potential. To perform the AF analysis, samples of Brazil nuts were treated separately. The AF from the shell and kernel were extracted by chloroform and analysed by the HPLC-FD system in isocratic mode. The Aspergillus section Flavi count was 21.67% lower. The production of AF by the isolated fungi was 30% for sanitised and 23.8% for non-sanitised samples. The concentrations obtained of AFB₁ and AFG₁ were higher than those of AFB₂ and AFG₂. The AFB₁ concentrations of shelled nuts and shell samples were 35.0 and 1.78 μg/kg, respectively. AFB₂ and AFG₂ were detected only in shelled nut samples. The HPLC-FD analysis presented limits of detection (LOD) and quantification (LOQ) of 0.2 and 0.4 μg/kg, respectively.     

Ochratoxin A production by Aspergillus carbonarius on some grape varieties grown in Italy

Fungi responsible for the presence of Ochratoxin A (OTA) in grapes have been identified as belonging to the black aspergilli, Aspergillus section Nigri, among which Aspergillus carbonarius is the main producer. Despite the widespread occurrence of OTA in various types of wine, there is no specific information on the ability of black aspergilli to infect berries and to produce OTA in different grape varieties. The aim of this study was to investigate the susceptibility to A carbonarius of twelve grape varieties representative of a wide range of characteristics, including geographical distribution. In all the conditions of berry status (intact and damaged) and temperature (20 and 25 °C) tested, the incidence of colonised berries and their OTA contents were always low in ‘Bianco d'Alessano’, ‘Pampanuto’ and ‘Uva di Troia’. In contrast, ‘Cabernet Sauvignon’, ‘Trebbiano’ and ‘Verdeca’ showed high fungal incidence and OTA content. The information gathered is relevant for defining risk factors for OTA presence in grapes, even if these data need to be confirmed by observations in vineyards. Copyright © 2004 Society of Chemical Industry     

Co-occurrence of aflatoxins,ochratoxin A and zearalenone in Capsicum powder samples available on the Spanish market

The aim of this study was to determine the co-occurrence of aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) in paprika and chilli samples purchased in Spain, using HPLC with fluorescence detection. The occurrence of mycotoxin in 64 paprika samples was 59% for AFs, 98% for OTA and 39% for ZEA, whereas in the 35 chilli samples, the contamination was 40% for AFs, 100% for OTA and 46% for ZEA. None of the samples had AFs levels higher than the legally allowable limits. Regarding the co-occurrence of mycotoxins, 75% of paprika samples and 65% of chilli samples contained more than one mycotoxin. Chilli samples generally had lower concentrations of AFB1, AFB2, total AFs and OTA than had paprika samples. The high incidence of OTA contamination suggests that additional legislation may be required to for these kinds of spices.     

Multiplex Real‐Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera

Abstract: Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real‐time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene‐producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON‐ and OTA‐producing fungi can be detected and quantified in a single reaction tube using this multiplex real‐time PCR method. Practical Application: This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal‐based foods.     

Validation of the procedure for the simultaneous determination of aflatoxins ochratoxin A and zearalenone in cereals using HPLC-FLD

Method validation for quantitative analysis of aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) in cereals using HPLC with fluorescence detector (FLD) is described. Mycotoxins were extracted with methanol?:?water (80?:?20) and purified with a multifunctional AOZ immunoaffinity column before HPLC analysis. The validation of the analytical method was performed to establish the following parameters: specificity, selectivity, linearity, limits of detection (LOD) and quantification (LOQ), accuracy, precision (within- and between-day variability), stability, robustness, measurement of performance, and measurement of uncertainty. Calibration curves were linear (r?>?0.999) over the concentration range, from the LOQ to 26, 40 and 400?ng/g for AFs, OTA and ZEA, respectively. LOD and LOQ were 0.0125 and 0.05?ng/g for aflatoxin B1 (AFB1) and G1 (AFG1), 0.0037 and 0.015?ng/g for aflatoxin B2 (AFB2) and G2 (AFG2), as well as 0.05 and 0.2?ng/g for OTA and 0.5 and 2?ng/g for ZEA, respectively. The mean recovery values were 77–104% for different concentrations of AFs, OTA and ZEA in spiked cereal samples. Both intra- and inter-day accuracy and precision were within acceptable limits. This method was successfully applied for the simultaneous determination of mycotoxins for 60 cereal samples collected from Malaysian markets. Fifty per cent of the cereal samples were contaminated with at least one of these mycotoxins, at a level greater than the LOD. Only one wheat sample and two rice samples were contaminated with levels greater than the European Union regulatory limits for AFs and OTA (4 and 5?ng/g). The means and ranges of mycotoxins obtained for the cereal samples were 0.4?ng/g and 0.01–5.9?ng/g for total AFs; 0.18?ng/g and 0.03–5.3?ng/g for OTA; and 2.8?ng/g and 2.4–73.1?ng/g for ZEA, respectively. The results indicate that the method is suitable for the simultaneous determination of AFs, OTA and ZEA in cereals and is suitable for routine analysis.