Aflatoxins contamination in spices and processed spice products commercialized in Korea

A survey for total aflatoxins (aflatoxins B₁, B₂, G₁, and G₂) was conducted on 88 spices and processed spice products commercialized in Korea. The presence of aflatoxins was determined by high-performance liquid chromatography (HPLC) with fluorescence detector using immunoaffinity column clean-up. Total aflatoxins (AFs) are detected in 12 samples (13.6% of incidence) including seven red pepper powder, two red pepper pastes (Kochujang), two curry and one ginger product. The contamination levels are 0.08–4.45 μg/kg as aflatoxin B₁ and 0.08–4.66 μg/kg as AFs. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis on contaminated samples was conducted for the confirmation of detected aflatoxins. The 12 samples which showed aflatoxins by HPLC/FLD were confirmed as aflatoxins by LC–MS/MS.     

Development of a class-specific monoclonal antibody-based ELISA for aflatoxins in peanut

Three class-specific monoclonal antibodies against aflatoxins were screened by a designed strategy in which aflatoxin G₂ was used as competitor in the screening ELISA system. With a high cross-reactivity (65%) to aflatoxin G₂, antibody 10C9 had the most similar sensitivity for five aflatoxins (AFB₁, AFB₂, AFG₁, AFG₂ and AFM₁), whose I₅₀ values were in a range of 2.1–3.2 ng ml⁻¹. So, antibody 10C9 was selected to develop an ELISA for determination of aflatoxin B₁, B₂, G₁, G₂ and total of them in peanut samples. And spiked recoveries were from 87.5% to 102.0%. The results indicate that the ELISA developed can accurately determine total aflatoxins in samples of peanuts after the simple and rapid extraction procedure.     

High performance liquid chromatographic determination of aflatoxins in chilli,peanut and rice using silica based monolithic column

A simple and rapid high performance liquid chromatographic with fluorescence detection method for the determination of the aflatoxin B₁, B₂, G₁ and G₂ in peanuts, rice and chilli was developed. The sample was extracted using acetonitrile:water (90:10, v/v%) and then purified by using ISOLUTE® multimode solid phase extraction. After the pre-column derivatisation, the analytes were separated within 3.7 min using Chromolith® performance RP-18e (100–4.6 mm) monolithic column. To assess the possible effects of endogenous components in the food items, matrix-matched calibration was used for the quantification and validation. The recoveries of aflatoxins that were spiked into food samples were 86.38–104.5% and RSDs were <4.4%. The method was applied to the determination of aflatoxins in peanut (9), rice (5) and chilli (10) samples. Liquid chromatography–tandem mass spectrometry analysis using triple quadruple analyser and operated in the multiple reaction monitoring modes on the contaminated samples was performed for confirmation.     

Survey of roasted street-vended nuts in Sierra Leone for toxic metabolites of fungal origin

Consumption of mycotoxin contaminated foodstuffs is common in regions where foods are not adequately controlled and routinely monitored, and this could have adverse effects on the health of consumers. In this study, 100 samples of roasted nuts (50 cashew nuts and 50 peanuts) vended within two cities of Sierra Leone were analysed for mycotoxins and other microbial metabolites by a LC-MS/MS method. The peanut samples contained detectable levels of 17 microbial metabolites, including aflatoxins B₁, B₂, G₁ and G₂ and alternariol, while none of these metabolites were found in the cashew samples. Aflatoxins (max: 5,729 μg/kg; mean: 487.8 μg/kg) and alternariol (3 μg/kg) were found in 24% and 2% of the peanut samples, respectively. One-third of the aflatoxin-contaminated peanut samples contained aflatoxins at levels exceeding the total aflatoxin limit of 4 μg/kg set by the European Union. Aflatoxin contamination of Sierra Leonean peanuts is high and requires urgent intervention to reduce consequent exposure.     

Determination of aflatoxins in peanut butter and sesame samples using high-performance liquid chromatography method

In this study, total number of samples analysed were 20 packages of sesame and 20 cans of peanut butter, which were collected from Ankara local markets, Turkey. Extraction and determination of aflatoxins have been made by immunoaffinity column technique and high-performance liquid chromatography (HPLC) method. Mean levels (±SE) of aflatoxins B₁, B₂, G₁ were found to be 15.756±3.129 ng/g, 1.232±0.244 ng/g and 9.689±1.005 ng/g, respectively in peanut butter samples. Regarding the sesame samples, mean level of aflatoxin G₁ was found to be 0.754±0.213 ng/g. Our data revealed that while aflatoxin levels found in sesame samples were within the Turkish Food Codex (TFC) values, for peanut butter samples, they were higher than the TFC values.     

Single-Laboratory Validation for the Determination of Aflatoxin B1, B2, G1, and G2 in Foods Based on Immunoaffinity Column and Liquid Chromatography with Postcolumn Derivatization and Fluorescence Detection

This study presents a method validation procedure for the determination of aflatoxin B₁, B₂, G₁, and G₂ in hazelnut, hazelnut paste, walnut, peanut, pistachio, corn, and wheat. The method consisting of clean-up with immunoaffinity column, high performance liquid chromatography with postcolumn derivatization and fluorescence detection was validated in accordance with Commission Regulation 2004/882/EC. The selectivity, linearity, decision limit, detection capability, detection and quantification limits, precision, recovery, ruggedness, and measurement uncertainty of the method were determined. The limit of detection and limit of quantification values (μg/kg) were: aflatoxin B₁, 0.02, 0.07; aflatoxin B₂, 0.01, 0.02; aflatoxin G₁, 0.02, 0.07; and aflatoxin G₂, 0.01, 0.03. The relative standard deviation values for the repeatability and within-laboratory reproducibility were below 4 and 5 %, respectively. The recovery values of the spiked samples ranged from 80 to 105 %. These results complied with minimum performance criteria established by regulation 2006/401/EC. Therefore, the procedure can be implemented for the routine analysis of aflatoxins in the studied matrices.     

Distribution of fungi and aflatoxins in a stored peanut variety

The objective of the present study was to evaluate the mycoflora and occurrence of aflatoxins in stored peanut samples (hulls and kernels) from Tupã, State of São Paulo, Brazil. The samples were analyzed monthly over a period of one year. The results showed a predominance of Fusarium spp. (67.7% in hulls and 25.8% in kernels) and Aspergillus spp. (10.3% in hulls and 21.8% in kernels), and the presence of five other genera. The growth of Aspergillus flavus was mainly influenced by temperature and relative humidity. Analysis of hulls showed that 6.7% of the samples were contaminated with AFB₁ (mean levels = 15–23.9 μg/kg) and AFB₂ (mean levels = 3.3–5.6 μg/kg); in kernels, 33.3% of the samples were contaminated with AFB₁ (mean levels = 7.0–116 μg/kg) and 28.3% were contaminated with AFB₂ (mean levels = 3.3–45.5 μg/kg). Analysis of the toxigenic potential revealed that 93.8% of the A. flavus strains isolated were producers of AFB₁ and AFB₂.     

Aflatoxins in nuts assayed by immunological methods

 Different kinds of raw and processed nuts available in the local retail market were investigated for aflatoxin content. Total aflatoxins and aflatoxin B₁ content were determined by immunoaffinity chromatography with fluorescence detection after reaction with bromine solution and by immunoenzymatic test kits. Of 29 investigated samples, 38% were contaminated. The total aflatoxin content in contaminated samples was between 1.20 μg/kg for peanuts and 5.50 μg/kg for walnuts. The concentration of aflatoxin B₁ found in contaminated samples was between 0.35 μg/kg for cashew nuts and 4.04 μg/kg for walnuts. The mean recovery of total aflatoxins was 95% for the Ridascreen test and 92% for immunoaffinity chromatography with fluorescence detection. For aflatoxin B₁ the mean recovery was 84%. Received: 4 March 1999 / Revised version: 17 May 1999     

Detection of Aspergillus spp. and aflatoxin B1 in rice in India

Twelve hundred rice samples consisting of paddy (675) and milled rice (525) were collected from 20 states across India. These samples were assessed for Aspergillus spp. infection on selective medium and aflatoxin B₁ (AFB1) by indirect competitive ELISA. In this investigation, Aspergillus flavus contamination dominated in all the seed samples. The other major contaminants were Aspergillus niger, Aspergillus ochraceus and Aspergillus parasiticus. Out of 1200 rice samples, 67.8% showed AFB1 ranging from 0.1 to 308.0 μg/kg. All the paddy samples from Chattishgarh, Meghalaya and Tamil Nadu showed AFB1 contamination. Milled rice grains from different states showed below the permissible levels of AFB1 (average 0.5–3.5 μg/kg). Eighty-two percent of samples from open storage that were exposed to rain showed AFB1 contamination followed by one-year-old seed. Out of 1200 samples, 2% showed AFB1 contamination above the permissible limits (>30 μg/kg). This is the first comprehensive report of aflatoxin contamination in rice across 20 states in India.     

Reduction of aflatoxins in peanut meal by extrusion cooking in the presence of nucleophiles

This study explored the feasibility of degrading aflatoxins in contaminated peanut meal by extruding in the presence of calcium chloride with lysine and methylamine. A 2 × 2 × 3 experiment (moisture, pH and nucleophile) was designed to screen for a nucleophile to use in a study of extrusion conditions to degrade aflatoxins in peanut meal. The nucleophile was mixed with peanut meal at 2 g/100 g protein level and the samples “spiked” with aflatoxin standards. They were extruded using a single screw extruder, and aflatoxins quantified by HPLC.The presence of calcium chloride impeded the degradation of aflatoxins by extrusion. However, methylamine and lysine showed comparable efficacies to mediate aflatoxin reduction. There were significant (p ≤ 0.05) interactions between moisture and pH, as well as moisture and temperature. Contour plots from regression models (R² = 0.85) showed a bimodal effect of moisture on aflatoxin degradation. Extrusion cooking reduced aflatoxins from an initial 417.72 μg/kg to 66.87 μg/kg (i.e. 84% reduction) in the peanut meal.     

Degradation of aflatoxins by extrusion cooking: Effects on nutritional quality of extrudates

Extrusion of artificially contaminated food is reported to degrade aflatoxins to varying degrees depending on the extrusion conditions. This work sought to determine the (1) efficacy of extrusion cooking in destroying naturally contaminated peanuts and (2) nutritional quality of decontaminated peanut meal. Naturally contaminated peanut meal was extruded by varying the moisture (20, 28, 35 g/100 g), pH (7.5, 9.5) and extruder die diameter (2.5, 3, 3.5, 4.0 mm). Aflatoxins levels in the extrudates were determined using HPLC procedures, and the nutritional quality was assessed using in-vitro methods. The highest aflatoxin reduction in naturally contaminated peanut meal was 59% at feed moisture content of 35 g/100 g. Higher (91%) reduction was achieved in the artificially contaminated peanut meal at moisture of 20 g/100 g. In-vitro protein digestibility and Fluorodinitrobenzene (FDNB)-available lysine of the extrudates were not significantly different from non-extruded peanut meal. Extrusion conditions for aflatoxin reduction did not adversely affect protein nutritional quality.     

Determination of ochratoxin A in organic and non-organic cereals and cereal products from Spain and Portugal

The objective of this work was to know the occurrence of OTA in organic and non-organic cereals and cereal products from Spain and Portugal. A method based on extraction with matrix solid phase dispersion (MSPD) using octylsilica (C₈) followed by liquid chromatography coupled with fluorescence detection (LC–FD) was used to determine OTA from the selected samples. Recoveries of OTA from the studied samples spiked at 10 ng/g level ranged from 78% to 89% with a standard deviation of 3.66. The limits of detection and quantification of this method were 0.05 and 0.19 ng/g, respectively. Furthermore, LC–FD after OTA methylation was used to confirm the identity of OTA in all positive samples. This procedure was applied to 83 organic and non-organic samples including rice, wheat, barley, rye, oats and maize from Spain and Portugal. OTA was detected in 22% of the samples, with concentrations ranging from 0.20 to 27.10 ng/g. From the total OTA contaminated samples (n = 18), 72% were organic cereal and 28% were non-organic cereal samples, with mean concentrations of 1.64 and 0.05 ng/g, respectively. The 66% and 34% of contaminated samples were from Spain and Portugal, respectively, with mean concentrations of 0.93 and 0.64 ng/g for each country. Six contaminated samples exceeded the maximum limits (ML) for OTA fixed by European Commission Regulation (5 μg/kg), among them three were from Spain and three from Portugal.     

SCREENING AND QUANTIFICATION OF AFLATOXINS AND OCHRATOXIN A IN DIFFERENT CEREALS CULTIVATED IN ROMANIA USING THIN-LAYER CHROMATOGRAPHY-DENSITOMETRY

ABSTRACT

The main focus of our study was to implement a rapid, inexpensive and reliable method that could be utilized to check the cereals for safety (i.e., screening for total aflatoxins, as well as individual B₁, B₂, G₁, G₂ aflatoxins and ochratoxin A). We developed a protocol by which we were able to isolate mycotoxins from cereals collected from different regions of Romania. After extraction in chloroform, the mycotoxins were separated by thin‐layer chromatography (TLC) and quantified using densitometry. Forty‐three samples of different cereals (wheat, maize, rye and Triticale) were analyzed. Twenty‐five of the 43 samples (58.14% of the total number) were found to be contaminated with different mycotoxins in various concentrations: aflatoxin B₁ (1.6–5.7 µg/kg), aflatoxin B₂ (0.89–4 µg/kg), aflatoxin G₁ (1.2–5.76 µg/kg), aflatoxin G₂ (0.96–3.4 µg/kg) and/or 4.3–30 µg/kg ochratoxin A. The concentration of total aflatoxin contamination ranged from 11.2 to 10.8 µg/kg. Among the different cereals, the highest number of contaminated samples was found to be in the wheat samples (62.5%). The method outlined in this study has been adopted already by our laboratory for current analyses of mycotoxins. The results obtained using this method is in compliance with the strict regulatory guidelines developed both in Romania, as well as in the European Union.

PRACTICAL APPLICATIONS

Thin‐layer chromatography (TLC) is a rapid, inexpensive and convenient method that can be used routinely to screen for mycotoxins. This article describes the detailed procedures for the extraction, purification and quantification of aflatoxins and ochratoxin A, using TLC. Using this method one can identify concomitantly five different mycotoxins and by coupling it with densitometry a quantitative determination is also possible. Therefore, this could become a routine technique in regional laboratories responsible for checking agrifood safety.     

Natural infection of cowpea (Vigna unguiculata (L.) Walp.) by toxigenic fungi and mycotoxin contamination in Benin, West Africa

Natural infection of cowpea by toxigenic fungi and mycotoxin contamination in Benin, West Africa were studied. Cowpea samples were collected at harvest (T₀) and after three months of storage (T₃) from the four agro-ecological zones of the country. A total of 92 representative samples were analysed for the two periods. About 23 fungal species were identified on cowpea seed samples across zones of which Aspergillus flavus, a fungus that produces aflatoxins, was most frequently encountered. Fusarium species shown to produce fumonisins were not recorded from cowpea seeds. Overall incidence of A. flavus infection was found to increase after storage from 7.6% at T₀ to 28.25% at T₃. In spite of this natural infection of cowpea, very low levels of fumonisin and aflatoxin were detected. Only three out of the 92 cowpea samples, all collected at T₀, were found to be fumonisin B₁ positive with a mean level of 0.03 μg/g. Similarly, only six samples out of the 92, all collected at T₃, were aflatoxin B₁ positive with mean levels of 3.58 μg/kg. Fumonisin (B₂ and B₃) and aflatoxin (B₂, G₁ and G₂) were not detected in any of the samples. Contrary to the situation with maize and groundnut where high levels of toxin are often detected in naturally infected samples, the current results indicate that cowpea is less susceptible to mycotoxin contamination. A low susceptibility could be due to the presence in cowpea of substances that inhibit mycotoxin biosynthesis. Further investigations are underway to confirm this hypothesis.     

The determination of vitamin D3 in bovine milk by liquid chromatography mass spectrometry

A renewed international interest in vitamin D status has revealed significant deficiencies in several populations, including Australia. Vitamin D exists in two forms, cholcalciferol (D₃) and ergocalciferol (D₂). The main source of vitamin D₃ is from exposure of 7-dehydrocholesterol present in the skin to UV irradiation. However, there is an absolute requirement for vitamin D through proper dietary intake if humans live in the absence of sunlight or exclusively indoors. Bovine milk is considered to be a good dietary source of vitamin D₃, even though the levels are quite low. This paper describes robust methods using liquid chromatography–linear ion trap mass spectrometry (LC–MSⁿ) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to measure the levels of vitamin D₃ in fresh bovine milk (0.05 μg/100 ml), commercial (natural and fortified) milk samples (0.01–2 μg/100 ml) and a dairy based infant formula (8 μg/100 g), without the need for extensive clean-up procedures. The limits of quantification (LOQ) are 0.01 μg/100 ml and 0.02 μg/100 ml for LC–MSⁿ and LC–MS/MS, respectively. Recoveries of vitamin D₃ added to the samples prior to saponification were satisfactory (range 60–90%). 25-Hydroxyvitamin D₃ was not present in any of the samples analysed (LOQ = 0.01 μg/100 ml, recovery range 30–40%).     

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.     

Comparison of homogenization techniques and incidence of aflatoxin contamination in dried figs for export

To determine differences in mean aflatoxin contamination and subsample variance from dry and slurry homogenizations, 10 kg of six different, naturally contaminated dried fig samples were collected from various exporting companies in accordance with the EU Commission Directive. The samples were first dry-mixed for 5 min using a blender and sub-sampled seven times; the remainder was slurry homogenized (1 : 1, v/v) and sub-sampled seven times. Aflatoxin B₁ and total aflatoxin levels were recorded and coefficient of variations (CV) computed for all sub-samples. Only a small reduction in sub-sample variations, indicated by the lower CV values, and slight differences in mean aflatoxin B₁ and total aflatoxin levels were observed when slurry homogenization was applied. Therefore, 7326 dried figs, destined for export from Turkey to the EU and collected during the 2008 crop year, were dry-homogenized and tested for aflatoxins (B_1, B_2, G₁ and G₂) by immunoaffinity column clean-up using RP-HPLC. While 34% of the samples contained detectable levels of total aflatoxins (0.20–208.75 µg kg^?1), only 9% of them exceeded the EU limit of 4 µg kg^?1 in the range 2.0–208.75 µg kg^?1, respectively. A substantial increase in the incidence of aflatoxins was observed in 2008, most likely due to the drought stress experienced in Aydin province as occurred in 2007.     

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products

A sensitive and specific monoclonal antibody (Mab) against aflatoxin M₁ (AFM₁), named as 2C9, was selected by semi-solid HAT medium. It exhibited high affinity for AFM₁ of 1.74 × 10⁹ L/mol and no cross-reactivity to aflatoxin B₁, B₂, G₁ and G₂. Based on the antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM₁ in milk and infant milk products. Assays were performed in the AFM₁-BSA coated (0.0625 μg/mL) ELISA format in which the antibody was diluted 1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assay performance were optimised. Finally, the limits of detection were 3 ng/L for milk and 6 ng/L for milk-based cereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged from 91% to 110%. Thirty samples were analysed, and concordant results were obtained when the data were compared with a reference high-performance liquid chromatography method.     

Simple and high-throughput method for the multimycotoxin analysis in cereals and related foods by ultra-high performance liquid chromatography/tandem mass spectrometry

A rapid, reliable and sensitive method was developed to determine 12 mycotoxins (deoxynivalenol, aflatoxins B1, B2, G1, G2 and M1, fumonisins B1 and B2, ochratoxin A, HT-2 and T-2 toxin and zearalenone) simultaneously in maize, walnuts, biscuits and breakfast cereals. The method is based on a single extraction step using acetonitrile/water mixture (80/20 v/v) followed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). The selectivity of the MS/MS detection allowed the elimination of further clean up steps. Extraction, chromatographic and detection conditions were optimised in order to increase sample throughput and sensitivity. Matrix-matched calibration was used for quantification and recoveries of the extraction process ranged from 70.0% and 108.4%, with relative standard deviations lower than 25% in all the cases, when samples were fortified at 5 and 50 μg/kg. Limits of detection ranged from 0.01 to 2.1 μg/kg and limits of quantification ranged from 0.03 to 6.30 μg/kg, which were always below the tolerance levels of mycotoxins set by European Union in the matrices evaluated. Several samples were analysed and aflatoxins B1, B2, G1, G2 and T-2 toxin were detected in one maize sample, with concentrations lower than 6.0 μg/kg and deoxynivalenol was detected in a breakfast cereal at 42.1 μg/kg.     

Fungal contamination and aflatoxin content of maize,moringa and peanut foods from rural subsistence farms in South Haiti

Mycotoxins are toxic, low molecular weight compounds produced by fungi. Among them, aflatoxins are the most carcinogenic and they mainly impact on rural communities of developing countries. The present study supplies data on mycobiota and aflatoxin contamination in the most common food products consumed in Haiti. The study concerns analyses performed on 49 samples of meals and seeds collected in South Haiti and tested for fungal occurrence and aflatoxin content by HPLC-DAD technique. The results revealed that three main fungal genera affected Haitian food products: Aspergillus spp. (Section Flavi and Nigri), followed by Penicillium spp. and Fusarium spp. Aflatoxin was present in more than half of the samples of maize (Zea mays L.) kernels (55%), maize meal (57%) and moringa (Moringa oleifera Lam.) seeds (64%), and in 25% of peanut (Arachis hypogaea L.) samples. The tested food products were mostly contaminated by aflatoxin B₁ (AFB₁) followed by aflatoxin B₂ (AFB₂), while no aflatoxins type G were detected. The total concentration of aflatoxins in the positive samples was 228 μg/kg on average, i.e., fifty-seven and eleven times higher than the maximum levels allowed in Europe and USA, respectively. Both the presence of aflatoxigenic fungi and aflatoxin contamination in maize kernels seemed to be related to agricultural practices, such as weed control, irrigation and growing cycle length. These findings suggest that the Haitian population is strongly exposed to aflatoxin risk. This risk could be reduced by exploiting simple and accessible farming strategies for minimizing mycotoxin contamination, at least for maize.