Aflatoxins in rice – A limited survey of products marketed in Austria

Eighty-one rice samples were purchased from different markets in Vienna and were analysed for their aflatoxin content. The samples were extracted using methanol in water (80/20 v/v) followed by immunoaffinity clean up. The determination was carried out by HPLC–FLD coupled to a Kobracell. Different samples including basmati rice, whole grain rice, long grain rice, short grain rice as well as puffed rice were investigated. Moreover, conventionally and organically produced rice were compared. The results revealed that 24 out of 81 samples contained detectable amounts of aflatoxins. Aflatoxin B₁ could be quantified in 15 samples and aflatoxin B₂ in one sample. The contamination range was noted to be between 0.45 μg kg⁻¹ and 9.86 μg kg⁻¹ for aflatoxin B₁ and 1.5 μg kg⁻¹ for aflatoxin B₂. Aflatoxins G₁ and G₂ were not detected in any sample. Three samples exceeded the maximum levels set in the European Union; having AFB₁ concentrations of 2.16, 2.85 and 9.86 μg kg⁻¹. In the three organic produced rice samples only traces of aflatoxins were found.     

Aflatoxigenic fungi and aflatoxins occurrence in sultanas and dried figs commercialized in Brazil

The presence of aflatoxins, Aspergillus flavus and Aspergillus parasiticus in dried fruits was investigated. A total of 62 dried fruit samples were analyzed (24 black sultanas, 19 white sultanas and 19 dried figs). A total of 10 A. flavus isolates were found, nine in one white sultana sample (corresponding to 18% infection) and one isolate in dried figs (2%), and all of them were aflatoxin B₁ and B₂ producers. A. parasiticus was not found. Aflatoxins were detected in 3 of 19 (16%) white sultana samples analyzed and, the limits were not higher than 2.0 μg/kg. In dried figs 11 of 19 (58%) samples were contaminated with aflatoxins and, with exception of one sample that was contaminated with 1500 μg/kg of B₁ aflatoxin, the others had less than 2.0 μg/kg. Neither aflatoxigenic or aflatoxins contaminated black sultanas.     

Regional monitoring plan regarding the presence of aflatoxin M1 in pasteurized and UHT milk in Italy

The aim of the study involved evaluation of the presence of aflatoxin M₁ in milk for sale in a specific North West Italian region, Piedmont. The study, conducted from November 2003 to July 2005, was linked to the specific emergency situation which arose due to the climatic conditions during the summer of 2003 which encouraged the development of aflatoxin B₁ in items used for animal feed. This in turn led to the transfer of the metabolite, aflatoxin M₁, into the milk. In total some 316 milk samples were collected during the commercial phase by the official control bodies and analysed. The analysis involved the use of high pressure liquid chromatography (HPLC) combined with fluorimetric measurement, and purification of the extracts using immunoaffinity columns. The results indicated only 2 non conforming samples (0.6%), with limits higher than those set out in the regulations (0.05 μg L⁻¹). In addition, the analyses revealed, in 5 samples (1.6%), threshold values of 0.05 μg L⁻¹. From the data obtained it can be seen that the “aflatoxins” problem only marginally affected Piedmont Region though the trend for average monthly values suggests a return to the use of contaminated animal feed as soon as official controls are less intensive.     

Occurrence of AFM1 in urine samples of a Brazilian population and association with food consumption

This survey evaluated the presence of AFM₁ in human urine samples from a specific Brazilian population, as well as corn, peanut, and milk consumption measured by two types of food inquiry. Urine samples from donors who live in the city of Piracicaba, State of São Paulo, Brazil were analyzed to detect the presence of aflatoxin M₁ (AFM₁), an aflatoxin B₁ metabolite, which may be used as aflatoxin B₁ exposure biomarker. The AFM₁ analysis was performed using immunoaffinity clean-up and detection by high-performance-liquid chromatography with fluorescence detector. A total of 69 samples were analyzed and 45 of them (65%) presented contaminations ⩾1.8 pg ml⁻¹, which was the limit of quantification (LOQ). Seventy eight percent (n = 54) of the samples presented detectable concentrations of AFM₁ (>0.6 pg ml⁻¹). The AFM₁ concentration among samples above LOQ ranged from 1.8 to 39.9 pg ml⁻¹. There were differences in food consumption profile among donors, although no association was found between food consumption and AFM₁ concentration in urine. The high frequency of positive samples suggests exposure of the populations studied to aflatoxins.     

Pressurized fluid extraction for quantitative recovery of aflatoxins B1 and B2 from pistachio

A pressurized fluid extraction method for the extraction of aflatoxins B₁ and B₂ from contaminated pistachio samples was developed using a modified supercritical fluid extractor. The parameters of temperature, pressure, and flow rate of the solvent were optimized for the extraction process. The pressure variation in the range of (10–100) bar had an insignificant effect on the aflatoxins extraction yield. Solution of 80% (v/v) methanol in water as the extraction solvent at flow rate of 0.5 mL/min and temperatures of higher than 80 °C were obtained for efficient extraction of aflatoxins B₁ and B₂ from spiked and naturally contaminated pistachio samples. The developed method in comparison with the AOAC method offered the repeatability (RSD) of 13.5% and 12%, respectively, while the extraction yield or recovery of the analytes was about 20% higher corresponding to those obtained with the AOAC method. The higher recovery of the developed method was validated by HPLC analysis and it was also applied to the peanut samples analysis.     

Aflatoxin contamination in edible nuts imported in Qatar

Edible nuts imported in Qatar from June 1997 to December 1998 were anlaysed for aflatoxins. Eighty-one nut samples were analysed in the second half of 1997 and contamination was detected in 19 samples with total aflatoxin level varied from a low of about 0.53 to a high of 289 μg/kg. Aflatoxin contamination was detected in pistachios and peanuts, while other nuts such as almond, cashew nut, walnut and hazel nut were found free from aflatoxins. During 1998 testing was carried out only for pistachios and about 101 samples were analysed; contamination was detected in 48 samples with total aflatoxin level in the range of 1.2–275 μg/kg. In pistachios without shell level of contamination was very high (total aflatoxin 8.3–275 μg/kg) compared to pistachio with shell (total aflatoxin 1.2–75 μg/kg). Aflatoxin B₁ and B₂ were detected in all the contaminated samples of pistachios, whereas aflatoxin G₁ and G₂ were detected only in three samples of pistachios at the level of 0.8–1.9 μg/kg for aflatoxin G₁ and 0.4–1.3 μg/kg for aflatoxin G₂.     

Natural occurrence of mycotoxins in cereals and spices commercialized in Morocco

Sixty samples of cereals (20 of corn, 20 of barley, and 20 of wheat) and 55 samples of spices (14 of paprika, 12 of ginger, 14 of cumin, and 15 of pepper) purchased from popular markets of Rabat and Salé in Morocco were analyzed for mycotoxins.Cereals samples were all analyzed for ochratoxin A (OTA). The average levels of contamination were 1.08, 0.42, and 0.17 μg/kg for corn, wheat, and barley, respectively. Samples of corn were also analyzed for zearalenone (ZEA) and fumonisin B₁ (FB₁) the average contaminations were 14 and 1930 μg/kg, respectively. Co-occurrence of OTA, FB₁, and ZEA was also checked. Spices samples were analyzed only for aflatoxins (AFs) and the average contaminations found for aflatoxin B₁ (AFB₁) were 0.09, 0.63, 2.88 and 0.03 μg/kg for black pepper, ginger, red paprika and cumin, respectively. The higher level of contamination was found in red paprika (9.68 μg/kg).The present report is the first one ever drafted on the natural co-occurrence of OTA, FB₁ and ZEA in cereals and on the occurrence of AFs in spices from Morocco.     

The potential of some spice essential oils in the control of A. parasiticus CFR 223 and aflatoxin production

Essential oils of sweet basil (Ocimum basilicum), cassia (Cinnamomum cassia), coriander (Coriandrum sativum) and bay leaf (Laurus nobilis) at 1–5% (v/v) concentration in palm kernel broth inoculated with spore suspension (10⁶/ml) of Aspergillus parasiticus CFR 223 were evaluated for their potential in the control of aflatoxigenic fungus A. parasiticus CFR 223 and aflatoxin production. Healthy sorghum grains (120/treatment) immersed in the oils and distributed in three petri dishes with wet cotton wool were also inoculated with spore suspension (10⁶/ml) of A. parasiticus CFR 223 and assayed for grain protection. Sweet basil oil at optimal protective dosage of 5% (v/v) was fungistatic on A. parasiticus CFR 223 and aflatoxins produced in vitro and on fungal development on sorghum grains (P ⩽ 0.05) with a residual effect that lasted for 32 days. In contrast, oils of cassia and bay leaf stimulated the mycelia growth of the fungus in vitro but reduced the aflatoxin concentration (B₁ + G₁) of the fungus by 97.92% and 55.21% respectively, while coriander oil did not have any effect on both the mycelia growth and aflatoxin content of the fungus. The combination of cassia and sweet basil oils at half their optimal protective dosages (2.5% v/v) completely inhibited the growth of the fungus. The feasibility of implementing the results of this study to control aflatoxins was examined by the addition of whole and ground dry basil leaves at 5% and 10% (w/w), respectively, to 10 g sorghum, groundnut, maize and melon seed after 35 days storage period. It was found that the addition of whole and ground basil leaves markedly reduced aflatoxin contamination; however, 10% (w/w) of whole leaves was more effective as the reduction in aflatoxin was between 89.05% and 91%.The findings showed that aflatoxins can be controlled by co-storing whole sweet basil leaves with aflatoxin infected foods. The economic value of the study lies in the simplified technique for control of aflatoxin contamination in agricultural products and the benefits derivable from the use of local resources.     

Mycotoxin occurrence in corn meal and flour traded in São Paulo,Brazil

In the present study, 60 samples of corn meal and flour traded in São Paulo were analysed for determination of aflatoxins and fumonisins B₁ (FB₁) and B₂ (FB₂). No aflatoxin was found in samples of both products. In corn meal, the concentrations of FB₁ and FB₂ ranged from 1.1 to 15.3 mg kg⁻¹ (mean: 5.2 mg kg⁻¹) and 0.2 to 3.9 mg kg⁻¹ (mean: 1.0 mg kg⁻¹), respectively. Corn flour presented lower levels of FB₁ (0.5–7.2 mg kg⁻¹; mean: 2.1 mg kg⁻¹) and FB₂ (0.1–1.8 mg kg⁻¹; mean: 0.7 mg kg⁻¹). Considering the average values of FB₁ found in corn meal samples, as well as food consumption estimates in Brazil, the worst case of FB₁ consumption would be 2.9 μg kg body weight⁻¹ per day. Results indicate the need for the adoption of practices to control the occurrence of fumonisins by manufacturers of corn products, mainly in corn meal.     

Fungal contamination and aflatoxin B1 of ‘egusi’ melon seeds in Nigeria

One hundred and thirty seven samples of melon seeds (Colocynthis citrullus L.) from randomly selected farmers’ stores in the humid forest and Northern Guinea savanna of Nigeria were analysed for the incidence of diseased seeds, moisture content, associated moulds and levels of aflatoxin B₁ contamination. The proportion of diseased seeds ranged from 2.5 to 37.3% in the forest and 2.1 to 17.9% in the savanna, while the seed moisture content varied from 5.3 to 10.4%, and 4.6 to 9.5% respectively. All the samples contained moulds, with the two genera, Aspergillus and Penicillium predominating, while A. flavus had the highest species count. The other common fungal isolates in order of decreasing incidence were A. niger, P. citrinum, Botryodiplodia theobromae, Cladosporium sp and A. clavatus. Thin layer chromatography analysis showed that 32% in the forest and 21% samples in the savanna contained aflatoxin B₁ with mean levels of 14.8 μg/kg in the forest and 11.3 μg/kg in the savanna respectively. Significant positive correlations were found between number of aflatoxin B₁ positive samples and the percentage of A. flavus infected samples and between the levels of diseased seeds and the levels of aflatoxin B₁ contamination.     

Aflatoxin B1 and ochratoxin A in breakfast cereals from athens market: Occurrence and risk assessment

A method for aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) determination in breakfast cereals is described using a simultaneous methanolic-aqueous extraction followed by immunoaffinity columns clean-up step and High Pressure Liquid Chromatography (HPLC) with Fluorescence Detector (FD). Recoveries were found to be 78% and 83% for AFB₁ and OTA, respectively, while the detection limit (DL) was 0.02 ng g^?1 for both mycotoxins. Both determinations were applied in fifty five samples of breakfast cereals purchased from Athens market. Results revealed the presence of AFB₁ in 56.3% of the samples examined (mean 1.42 ng AFB₁ g^?1). Seven samples (median 3.5 ng AFB₁ g^?1) were found to be contaminated at levels higher than the EU limit (2 g g^?1). OTA was detected in 60% of the samples (mean 0.18 ng g^?1). Nineteen samples were found to be contaminated by both mycotoxins. In addition in the present study the daily exposure to AFB₁ and OTA is discussed.     

Effect of gamma radiation on reduction of mycotoxins in black pepper

Gamma ray was applied to reduce mycotoxins, i.e. ochratoxin A (OTA) and aflatoxins B₁, B₂, G₁ and G₂ (AFB₁, AFB₂, AFG₁ and AFG₂) in black pepper. Response surface methodology (RSM) was applied to evaluate the effect of dose of gamma ray ranging from 0 to 60 kGy and mycotoxin concentration ranging from 10 to 100 ng g⁻¹ on the mycotoxin reduction. The maximum reduction was found at 60 kGy which was 52%, 43%, 24%, 40% and 36% for OTA, AFB₁, AFB₂, AFG₁ and AFG₂, respectively. Results showed the gamma rays even at 60 kGy were not effective in completely destroying of ochratoxin and aflatoxins.     

Aflatoxin-producing Aspergillus spp. and aflatoxin levels in stored cassava chips as affected by processing practices

Cassava chips (cassava balls, and cassava pellets) are derived cassava products traditionally produced by farmers in sub-Saharan Africa following fermentation, and drying of fresh roots of cassava, and are widely consumed in Cameroon. Once produced, this food commodity can be stored for more than two months and contaminated by a wide array of harmful microbes. In order to assess persistence of toxigenic fungi in cassava chips, aflatoxin-producing fungi (Aspergillus flavus, Aspergillus nomius, and Aspergillus parasiticus) and aflatoxins were contrasted at regular intervals in home-stored cassava chips collected in two locations of southern Cameroon throughout a two-month monitoring period. Three hundred and forty-six isolates of aflatoxin-producing fungi were found to be associated with all samples. A. flavus contaminated more samples in both types of chips (267 isolates in 53 samples), followed by A. nomius (58 isolates in 15 samples), whereas A. parasiticus was rarest. A direct competitive Enzyme-linked immunosorbent assay (ELISA)-based method was implemented to quantify the content in aflatoxins. Eighteen of the samples contained some aflatoxins at detectable levels whereas 54 did not. The levels of aflatoxin ranged between 5.2 and 14.5 ppb. The distribution of aflatoxin in positive samples depended on 8 parameters including pH, moisture content, storage duration, types of chips, level of contamination by aflatoxin-producing fungi, processing practices and storage facilities. From analysis of variance results, only pH (p < 0.01), duration of storage (p < 0.01), population of aflatoxin-producing species (0.0001) and the chip type (p < 0.05) were significantly related to aflatoxin in positive samples. A stepwise regression analysis (forward selection procedure) indicated that aflatoxin levels were significantly (p < 0.01) correlated with processing practices, storage facilities, and storage duration of the chips.     

Degradation of aflatoxins in aqueous buffer in the presence of nucleophiles

This study compared the efficacies of lysine, glycine and methylamine to mediate aflatoxin destruction in heated aqueous phosphate buffers. A 3 × 3 × 4 factorial design (buffer pH, heating temperature and nucleophile) was followed. The buffer was artificially contaminated (“spiked”) with mixed aflatoxin standard and heated for specified time periods. After the heat treatment residual aflatoxins were determined using HPLC procedures.Aflatoxin reduction was influenced by the buffer pH and the pK_a of the added nucleophile. When phosphate buffer (at pH 9) was heated to 150 °C the degradation rate constants (k) aflatoxin in the presence of lysine, glycine and methylamine were 0.11, 0.09 and 0.08 min⁻¹ respectively. Addition of calcium chloride to the “spiked” buffer lowered aflatoxin reduction, but upon adding lysine or methylamine, aflatoxin reduction was restored to some extent. These findings demonstrate the potential capability of lysine to mediate aflatoxin reduction.     

Validation of the procedure for the determination of aflatoxin B1 in animal liver using immunoaffinity columns and liquid chromatography with postcolumn derivatisation and fluorescence detection

The validation of the procedure for the determination of aflatoxin B₁ in animal liver (pig, chicken, turkey, beef, calf) was performed. The limit of detection (LOD) and limit of quantification (LOQ) were 2 ng/kg and 7.8 ng/kg, respectively. The repeatability of measurements, represented by the standard deviation (RSD_r) was 7.5%, 7.1%, and 4.8% at the contamination levels of 0.025 μg/kg, 0.050 μg/kg, and 0.075 μg/kg, respectively. The relative standard deviation for the within-laboratory reproducibility (RSD_R) was 18% at the level of 0.025 μg/kg and 22% at the levels of 0.050 μg/kg and 0.075 μg/kg. The measurement uncertainties at the same contamination levels were ±0.007 μg/kg, ±0.016 μg/kg, and ±0.023 μg/kg, respectively. The mean recovery was 72.8%, the decision limit (CCα) 0.063 μg/kg and the detection capability (CCβ) 0.080 μg/kg. The results indicate that the procedure is suitable for the determination of aflatoxin B₁ in animal liver and can be implemented for the routine analysis.     

Determination of Aflatoxin B1 levels in powdered red pepper

Insufficient hygiene conditions during drying, transport and storage stages in the production of red pepper could cause microbiological and mycological growth which could result in the formation of mycotoxins. This study was designed to assess the aflatoxin B₁ levels in 100 samples of powdered red pepper randomly obtained from markets in Istanbul using microtitre plate Enzyme Linked Immunosorbent Assay (ELISA). Aflatoxin B₁ levels were below the minimum detection limit (0.025 μg/kg) in 32 samples, between 0.025 and 5 μg/kg in 50 samples, whilst 18 samples had unacceptable contamination levels higher than the maximum tolerable limit (5 μg/kg), according to the Turkish Food Codex and the European Commission.     

Development of an immunochromatographic assay for detection of aflatoxin B1 in foods

A rapid, signal step and sensitive immunochromatographic (IC) test for detection of aflatoxin B₁ (AFB) in foods was described. Colloidal gold-labeled polyclonal antibody specific to AFB was used as the marker; AFB–BSA conjugate was the competitive antigen and goat-anti-rabbit antibody as control. Conditions for the analysis of AFB to be completed in less than 10 min were optimized. With visual observation, the lower limit was found to be around 2.5 ppb. For quantitative analysis by adopting a photometric strip reader, the lower detection limit was found to be around 0.05–0.1 ppb. The detection limit for AFB in food extract was around 2 μg/kg (ppb). The analytical recoveries for AFB added to extracts of rice, corn, and wheat and spiked directly to these foods range in 2–50 ppb were found to be 80.79% (CV, 10.92%) to 110.56% (CV, 7.95%). The CVs of all the assays were between 6.27% and 14.63%. Sixty seven naturally contaminated food samples were subjected to both IC strip and commercial ELISA kit test for AFB. Results showed a good correlation between these two methods with a square of correlation coefficient of 0.93 and slop of 1.13.     

A study on the occurrence of aflatoxin M1 in Iranian Feta cheese

This study was undertaken to determine the presence and levels of aflatoxin M₁ (AFM₁) in cheeses produced by different plants in the province of Tehran. For this purpose, a total of 80 cheese samples analyzed, and thin layer chromatography (TLC) was used to determine the presence and levels of AFM₁.AFM₁ was found in 82.5% of 80 of the cheese samples examined. The range of contamination levels varied among different months. AFM₁ in May, August, November, February samples ranged from 0.17 to 1.30, 0.15 to 2.41, 0.16 to 1.11, and 0.19 to 2.05 μg/kg, respectively, while the mean values were 0.41, 0.35, 0.36, and 0.52 μg/kg, respectively.The highest mean concentration of aflatoxin M₁ (AFM₁) was registered in February samples (0.52 μg/kg). The lowest mean concentration of aflatoxin M₁ was registered in August samples (0.35 μg/kg).Statistical evaluation showed that there were not significant differences (P > 0.05) between the concentrations of AFM₁ of cheese samples taken in May and August with November and February. In other words, AFM₁ contents of cheese samples taken in November and August were not lower than cheese samples taken in May and February. Almost 60.6% of the contaminated samples exceeded the maximum acceptable levels (0.25 μg/kg) that accepted by some of the countries such as Turkey.It was therefore concluded that, high occurrence of AFM₁ in cheese samples were considered to be possible hazards for human health.     

Survey of aflatoxin B1 in helva,a traditional Turkish food,by TLC

A total of 102 helva samples consisting of 34 plain helva, 34 helva containing cacao, and 34 helva containing pistachio nuts purchased from helva-factories and supermarkets in Adana of Turkey were analysed for aflatoxin B₁ (AFB₁) by thin-layer chromatography. The detection limit of AFB₁ was 1 μg kg⁻¹. Recovery experiments were carried out with spiked samples in the range 2–10 μg kg⁻¹ of AFB₁. No AFB₁ was found in any plain helva and helva containing cacao samples. On the other hand, of 34 helva containing pistachio nuts AFB₁ was determined in eight samples. AFB₁ was found in excess of Turkish legal limit of 5 μg kg⁻¹ in 4 of 102 helva samples. This paper reports the data of the first survey for the presence of AFB₁ in helva in Turkey.     

Investigations on the essential oil of Lippia rugosa from Cameroon for its potential use as antifungal agent against Aspergillus flavus Link ex. Fries

Lippia rugosa essential oil was tested for its effectiveness against Aspergillus flavus on artificial growth media. The chemical composition of the oil was determined by gas chromatography–mass spectrometry (GC–MS). Geraniol (51.5%), nerol (18.6%) and geranial (10.4%) were the main components of Lippia oil. After 8 days of incubation on essential oil supplemented medium, mycelium growth of A. flavus was totally inhibited by 1000 mg l^?1 of L. rugosa essential oil. The effect of essential oil on aflatoxin B₁ synthesis was evaluated in SMKY broth. The medium supplemented with different essential oil concentrations, was inoculated with A. flavus mycelium and incubated at 25 °C. After 2, 4, 6 and 8 days, aflatoxin B₁ (AFB₁) was quantified in the supernatant using Enzyme Linked Immuno-Sorbent Assay (ELISA). Results showed that aflatoxin B₁ synthesis was inhibited by 1000 mg l^?1 of L. rugosa essential oil after 8 days of incubation. The effect of the EO on the H⁺-ATPase pumping membrane was also evaluated in the presence of several concentrations of oil (200–2000 mg l^?1) by monitoring glucose-induced acidification of the external medium. L. rugosa essential oil at the concentration of 2000 mg l^?1 completely inhibited the activity of this enzyme. These data suggest that the essential oil of L. rugosa is a fungicidal for A. flavus and its possible cellular target include the H⁺-ATPase.Results obtained in the present study indicate the possibility of exploiting Lippia rugosa essential oil in the fight against strains of A. flavus responsible for biodeterioration of stored foods products.