Aflatoxin production by Aspergillus flavus in rumen liquor and its implications

Cow milk in infant and human nutrition is very significant. However, contamination of milk with aflatoxins is considered as a potential risk for human health. Aflatoxin is one of the major etiological factors in the development of hepatocellular carcinoma and is also found in the milk of lactating animals which could have consumed it through contaminated feedstuffs. Thus, exploration to isolate and identify the pathogenic microbe present in the rumen liquor were carried out. The screened fungal organism was identified as Aspergillus flavus by phenotypic (morphology and extrolite profiles) and molecular (β-tubulin gene sequences) characters. Fungal toxin was extracted using immuno-affinity column (IAC) and quantified by High Performance Liquid Chromatography (HPLC). The organism had potential to grow under aerobic and anaerobic conditions and also produce aflatoxin B_1. The aflatoxin B₁ production under aerobic condition was 0.902 ± 0.08 μg/ml culture broth and anaerobic condition was 0.925 ± 0.2 μg/ml culture broth. Aflatoxin B₂ was more compared to aflatoxin B₁ and the quantity was 14.472 ± 1 under aerobic condition and 1.467 ± 0.3 under anaerobic condition. The rumen liquor from which the isolation was carried out also showed the presence of aflatoxin B₁ (3.964 ± 0.5 μg/ml) and B₂ (1.170 ± 0.6 μg/ml). However, aflatoxin G₁ and G₂ were not present. Hence, the study suggests the ability of microbial ecosystem present inside the rumen to produce aflatoxin. This report on the aflatoxin production under aerobic and anaerobic conditions provides insights about the possibility of aflatoxin in cow milk thereby effecting human health. It is vital to reduce exposure of milking animals to contaminated moldy feed and take precautions to prevent fungal contaminations in the feed.     

Mycotoxin contamination in corn smut (Ustilago maydis) galls in the field and in the commercial food products

Corn infected with Ustilago maydis, causal agent of common smut disease, produces galls that are used as food in certain cultures, but may be contaminated with mycotoxins. The objective of this study was to determine mycotoxin levels in common smut galls (CSGs) collected from the field at corn ear reproductive stages R1 through R5 and in commercial CSGs products. The study was conducted in 2012 and 2013. A simple extraction method for five mycotoxins was devised and the results showed the presence of these compounds in CSGs in corn during ear development at various physiological stages. Fumonisin was the major mycotoxin in CSG samples in both 2012 (63%, ≤150.7 μg g⁻¹) and 2013 (46.9%, ≤20.9 μg g⁻¹); followed by aflatoxin (2012: 2%, ≤14.7 ng g⁻¹; 2013: 30.6%, ≤10.8 ng g⁻¹) and zearalenone (2012: ≤41.70 ng g⁻¹; 2013: ≤12.40 ng g⁻¹). Deoxynivalenol (DON) was only detected in 2012 (≤1.6 μg g⁻¹), and cyclopiazonic acid was only detected in 2013 (≤3.18 μg g⁻¹). Commercial canned and fresh CSG samples also contained detectable amounts of mycotoxins including aflatoxin, fumonisin, CPA, and DON. Aspergillus flavus was isolated from selected 2013 CSG field samples at R2 or older (0–1.6 × 10⁶ cfu/g), whereas Fusarium spp were isolated at R1 or older (0–7.5 × 10⁷ cfu/g). These results indicate that CSGs can be infected with mycotoxigenic fungi and contaminated with mycotoxins. The incidence of mycotoxins in commercially available CSG products was highly variable and warrants further study.     

Aflatoxins and ochratoxin A in dried eggplant and green bell pepper

In this study, 50 dried eggplant and 50 dried green bell pepper samples were analyzed in terms of their aflatoxin and ochratoxin A (OTA) content. Aflatoxins G₂, G₁, B₂, and B₁, and OTA contents were analyzed using high-performance liquid chromatography with a flame ionization detector (HPLC–FID). Total aflatoxin and, as well as aflatoxin G₂, G₁, B₂, and B₁ content in dried eggplant samples were ranged between 0.82 and 2.58, 0.10–0.23, 0.32–1.35, 0.12–0.67, and 0.17–0.71 μg kg⁻¹, respectively. Total aflatoxin and, as well as aflatoxin G₂, G₁, B₂ and B₁ content in dried green bell pepper samples were 0.81–2.42, 0.11–0.22, 0.32–1.38, 0.13–0.66, and 0.18–0.91 μg kg⁻¹, respectively. OTA content was varied from 8.88 to 21.35 μg kg⁻¹ in eggplant samples, and from 15.38 to 24.70 μg kg⁻¹ in dried green bell pepper samples. Of the dried eggplant samples and dried green bell pepper samples, 36% and 24% of them, respectively, had aflatoxin B₁ values which were below the minimum limit of detection (LOD) of 0.05 μg kg⁻¹. None of the analyzed samples exceeded the legal limit values of 10 μg kg⁻¹ for total aflatoxin content, and 5 μg kg⁻¹ for aflatoxin B₁ content. However, 80% of the dried eggplant samples and 100% of the dried green bell pepper samples exceeded the legal limit value for OTA content (15 μg kg⁻¹). According to the results, it was concluded that dried vegetables should be examined in terms of their aflatoxins. It is essential to analyze OTA content more thoroughly, as it has the potential to pose a risk for public health, as well as for the economy.     

Occurrence of four mycotoxins in cereal and oil products in Yangtze Delta region of China and their food safety risks

A total of 76 cereal and oil products collected from Yangtze Delta region of China were analyzed for occurrences of aflatoxins (AFs), aflatoxin B₁ (AFB₁), ochratoxin A (OTA), deoxynivalenol (DON) and zearalenone (ZEN). The mycotoxins were determined by the standard detection procedures using immunoaffinity column clean-up coupled with fluorometer (or HPLC-UV). ZEN was the most prevalent toxin, with the incidence of 27.6% (range = 10.0–440.0 μg kg⁻¹), and 9.2% of the evaluated samples were contaminated with a concentration higher than that of the legislation limit of China (60 μg kg⁻¹). AFs and AFB₁ were detected in 14.5% of the samples analyzed, the concentrations ranging 1.1–35.0 μg kg⁻¹ for AFs, and 1.0–32.2 μg kg⁻¹ for AFB₁; 4.0% of the samples had the concentrations of AFs and AFB₁ higher than that of the corresponding legislation limits of China (5.0, 10.0 and 20.0 μg kg⁻¹ for different products). OTA was detected in 14.5% of the cereal and oil products collected; the concentrations ranged 0.51–16.2 μg kg⁻¹. Only 2 samples showed OTA levels higher than that of the legislation limit of China (5.0 μg kg⁻¹). DON was detected in 7.9% of the samples; the concentrations ranged 100–700 μg kg⁻¹, and none of the samples showed DON concentration higher than that of the legislation limit of China (1.0 mg kg⁻¹). A total of 15.8% cereal and oil products were contaminated with at least two mycotoxins (multiple contaminations with different combinations including AFs-ZEN, AFs-OTA-ZEN, OTA-ZEN, ZEN-DON, OTA-ZEN-DON). The dietary exposure assessment results indicated that AFs (AFB₁), OTA, DON and ZEN from cereal-based products represented a series health risk to both adults and children in Yangtze Delta region of China. This is the first report of safety evaluation associated with major mycotoxins for the area.     

Effects of high pressure processing on immunoreactivity and microbiological safety of crushed peanuts

Peanuts are a common economical food source consumed worldwide but exist health concern of food allergy and are particularly susceptible to infection by the mold fungus Aspergillus flavus during storage, accumulating highly toxic substance aflatoxin. In this study, the effect of high pressure treatments on peanut immunoreactivity, peanut amino acid composition, A. flavus growth and aflatoxin contents on crushed peanuts was evaluated. Results showed that immunoreactivity of peanuts treated with 600 MPa and 800 MPa for 10 min was significantly lower (P < 0.05) than those of the control group by 69.2 ± 5.3% and 73.3 ± 1.9%, respectively. High pressure treatment at 800 MPa decreased total essential amino acid content as well as two nutritional indexes, the chemical score and the essential amino acid index, by 32.4 ± 2.1% and 31.1 ± 3.2%, respectively. The growth of aflatoxigenic fungi was inhibited in peanuts with aflatoxin accumulation that were subjected to different levels of pressure treatments during 30 days of storage. Peanuts treated with 600 MPa and 800 MPa had considerably lower aflatoxin levels, 0.26 μg/g and 0.22 μg/g in wet basis, respectively, than the control peanut aflatoxin level (9.08 μg/g) on day 30. Results were demonstrated that high pressure treatment had a significant inhibitory effect on A. flavus growth in peanuts and this contributes to reduction of aflatoxin production and accumulation instead of directly destroy aflatoxin. Taken together, the findings of this study indicated that high pressure treatment could preserve peanut quality by reducing food immunoreactivity and by eliminating A. flavus in peanuts.     

Occurrence of aflatoxin M1 in pasteurised,UHT milk and milk powder from goat origin

In the present study, 36 samples of pasteurised, ultra-high-temperature (UHT) treated and goat milk powder traded in the city of Campinas, Brazil, were analysed for aflatoxin M₁ (AFM₁), from October to December 2004 and March to May 2005. Results showed 25 (69.4%) positive samples for AFM₁ at levels of 0.011–0.161 μg L⁻¹ of milk, which were below the tolerance limit of 0.500 μg L⁻¹ as adopted for AFM₁ in milk by Brazilian regulations. Mean levels of AFM₁ in pasteurised, UHT and goat milk powder were 0.072 ± 0.048, 0.058 ± 0.044 and 0.056 ± 0.031 μg L⁻¹, respectively. It is concluded that the incidence of AFM₁ in goat milk traded in Campinas is high, but at levels that probably leads to a non-significant human exposure to AFM₁ by consumption of goat milks.     

Aflatoxin M1 in UHT milk consumed in Turkey and first assessment of its bioaccessibility using an in vitro digestion model

Contamination of milk and dairy products with aflatoxin M₁ (AFM₁) continues to receive increased attention because of its potential health hazard to humans. The first aim of this study was to know the occurrence and levels of AFM₁ in whole UHT milk from main processors in Turkey in order to make a preliminary exposure assessment. A total of 40 milk samples were analysed for AFM₁ using high performance liquid chromatography with fluorescence detection (HPLC-FD) after immunoaffinity column clean-up. Aflatoxin M₁ was detected in 20% of samples at levels ranging from <0.004 to 0.076 μg l⁻¹. Only two samples contained AFM₁ above the EU limit of  0.05 μg l⁻¹.The second aim of this study was to determine the bioaccessibility of AFM₁ from milk using an in vitro digestion model. The bioaccessibility of AFM₁ in spiked and naturally contaminated milk samples ranged from 80.5 to 83.8% and from 81.7 to 86.3%, respectively. No difference (P > 0.05) in AFM₁ bioaccessibility was found between spiked and naturally contaminated milk samples. This study also assessed the binding of AFM₁ by six probiotic bacteria under simulated gastrointestinal conditions. A 15.5–31.6% reduction in AFM₁ bioaccessibility was observed in the presence of probiotic bacteria. Based on the results obtained in the present study, the mean daily intake of AFM₁ through milk consumption was estimated as 0.008 ng kg⁻¹ b.w. day⁻¹ for Turkish adults.     

Aflatoxins occurrence through the food chain in Costa Rica: Applying the One Health approach to mycotoxin surveillance

Aflatoxins (AFs) are toxic metabolites produced by Aspergillus spp. and commonly found in crops, grains, feedstuff, and forages. Exposure to AFs has been associated with increased risk of liver cancer and growth retardation in humans, liver damage, immunosuppression, embryotoxicity in both animals and humans, and decreased milk, egg and meat production in animals. For the first time, the Costa Rican national mycotoxin surveillance programs for animal feed and food are considered as a whole, applying the One Health approach to the mycotoxin epidemiological research. Therefore, the aim of this study was to determine the occurrence of AFs in cereals, nuts, grains intended for animal and human consumption in Costa Rica.In animal feed and feed ingredients, 970 samples were analyzed for AFs from 2010 to 2016 with an overall prevalence of positive samples of 24.0% (ranging from 0.01 to 290 μg kg⁻¹). Only 2.5% of the samples failed to comply the regulation for total AFs (20 μg kg⁻¹ feed). From 5493 samples of agricultural commodities intended for human consumption analyzed from 2003 to 2015, there was an overall prevalence of AF positive samples of 10.8% (ranging from 0.48 to 500 μg kg⁻¹), and 2.8% did not comply the regulation for AFs (20 μg kg⁻¹). In both feed and food, the highest AF prevalence corresponded to corn ingredients (27.8%) and white corn (38.6%), respectively. Among the commodities intended for human consumption, red beans had the highest aflatoxin concentrations (500 μg kg⁻¹).     

Evaluation of fumonisin exposure by determination of fumonisin B1 in human hair and in Brazilian corn products

The aim of this study was to evaluate the dietary exposure to fumonisin B₁ (FB₁) through determination of residual FB₁ in hair and corn products consumed by 56 volunteers from Pirassununga and Erval Velho, Brazil. Data from FB₁ analyses in corn products and a Food Frequency Questionnaire (FFQ) were used for estimating the mean probable daily intake (PDI_M) for FB₁. FB₁ was detected in 4 human hair samples (7.2%), at a mean level of 21.3 ± 12.1 ng g⁻¹. The mean FB₁ level found in corn products was 360.4 ± 555.1 μg kg⁻¹. The PDI_M value of FB₁ in volunteers was 159 ± 47 ng kg⁻¹ body weight day⁻¹ which represents 7.9% of the provisional maximum tolerable daily intake (PMTDI) recommended for fumonisins. The FB₁ levels found in human hair samples from each volunteer were associated with their individual PDI of FB₁, indicating that exposure to FB₁ in the sample studied do not represent a health concern. This is the first report on the incidence of FB₁ in individual human hair in Brazil.     

Development of an aptasensor for the fast detection of Versicolorin A

Aflatoxins (AFs) are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. The molds may contribute to pre-harvest aflatoxin contamination of susceptible crops. For the customer and food producer, a predictive model for aflatoxin detection is very desirable. Versicolorin A (VerA), which is the first precursor in the pathway of aflatoxin B₁ (AFB₁) biosynthesis, shares similar toxic group with the furofuran structure in aflatoxin B₁. VerA exhibits a much lower teratogenic toxicity than AFB₁ and may be used as a predictive indicator for aflatoxin B₁ contamination of storage crops. Therefore, the development of a fast detection method for VerA is important. One of the randomly computer-generated aptamers of VerA was confirmed by isothermal titration calorimetry with K_d = 9.26 × 10⁻⁶ mol l⁻¹. In addition, a simple and sensitive label-free aptasensor was developed for the electrochemical detection of VerA. According to the results from differential pulse voltammetry (DPV), a linear relationship existed between the log conc. of VerA (ranged from 0.01 to 100 ng ml⁻¹) and the current (△I_p) with a limit of detection (LOD) of 10 pg ml⁻¹. The resulting aptasensor exhibited good reproducibility for detecting VerA and stability after storage for 15 days at 4 °C with acceptable anti-interference against ZEN, OTA, DON, and FB₁. When used in corn samples, the recoveries of VerA were determined to be in the range of 81.3%–104.4 %. Although with some intercross, result suggests that the obtained aptamer for VerA is potentially used as a sorbent for the preparation of solid-phase-extraction procedure to clean up food samples in conjunction with high-performance liquid chromatography analysis.     

Aflatoxin B1 in post-harvest peanuts and dietary risk in China

To monitor the aflatoxin contamination status in raw peanuts and evaluate the effect on public health, 1040 samples were collected from four agro-ecological zones throughout 12 provinces from 2009 to 2010 in China and then analyzed for aflatoxin B₁ (AFB₁) levels using High Pressure Liquid Chromatography (HPLC) and immunoaffinity columns. The results revealed that AFB₁ was detected in 25% of the samples, ranging from 0.01 to 720 μg/kg. The Monte Carlo and bootstrap methods were employed to estimate AFB₁ intake in children and adults and their potential liver cancer risk. The mean estimated intakes for children and adults were 0.218-0.222 ng/kg body weight (bw)/day and 0.106-0.108 ng/kg bw/day. The liver cancer risk, calculated by two approaches derived from the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and European Food Safety Authority (EFSA), were estimated at 0.003-0.17 cancer cases/year/100,000 and 24.7-1273 margins of exposure values, respectively. The results suggest that AFB₁ contamination in raw peanuts and dietary risk was low, but essential surveillance measures should be taken to protect public health.     

Multiple mycotoxin co-occurrence in maize grown in three agro-ecological zones of Tanzania

In this study, the co-occurrence of multiple mycotoxins in maize kernels collected from 300 households' stores in three agro-ecological zones in Tanzania was evaluated by using ultra high performance liquid chromatography/time-of-flight mass spectrometry (TOFMS) with a QuEChERS-based procedure as sample treatment. This method was validated for the analysis of the main eleven mycotoxins of health concern that can occur in maize: aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), aflatoxin G₁ (AFG₁), aflatoxin G₂ (AFG₂), ochratoxin A (OTA), deoxynivalenol (DON), fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), HT-2 toxin, T-2 toxin and zearalenone (ZEN). From each zone one major maize producing district for home consumption was chosen and 20 villages for each district were randomly selected for sampling. All mycotoxins of health concern, except for T-2 toxin, were detected in the maize samples. Particularly high levels of AFB₁ (50%; 3–1,081 μg kg⁻¹), FB₁ (73%; 16–18,184 μg kg⁻¹), FB₂ (48%; 178–38,217 μg kg⁻¹) and DON (63%; 68–2,196 μg kg⁻¹) were observed. Some samples exceeded the maximum limits set in Tanzania for aflatoxins or in European regulations for other mycotoxins in unprocessed maize. Eighty seven percent of samples were contaminated with more than one mycotoxin, with 45% of samples co-contaminated by carcinogenic mycotoxins, aflatoxins and fumonisins. Significant differences in contamination pattern were observed among the three agro-ecological zones. The high incidence and at high levels (for some) of these mycotoxins in maize may have serious implications on the health of the consumers since maize constitute the staple food of most Tanzanian population. Effective strategies targeting more than one mycotoxin are encouraged to reduce contamination of maize with mycotoxins.     

A survey of mycotoxins in random street-vended snacks from Lagos,Nigeria, using QuEChERS-HPLC-MS/MS

A survey in African snacks was carried out in order to evaluate the intake of 23 mycotoxins. The African snack samples were purchased from street vendors within Lagos metropolis (Nigeria) and evaluated for the presence of 23 mycotoxins using a modified QuEChERS procedure coupled with liquid chromatography-triple quadrupole linear ion trap mass spectrometer. The snacks included akara, baked coconut, coconut candy, donkwa, groundnut cake (kulikuli), lafun, milk curd (wara), fresh and dried tiger-nuts, and yam flour. Only three mycotoxins were detected in 23.8% of the studied snacks, and at concentrations ranging from 6 to 54 μg kg⁻¹. The concentrations of aflatoxin B₁ (AFB₁) and AFB₂ reached 23 μg kg⁻¹ and 3 μg kg⁻¹, respectively. Moreover a sample of baked coconut contained α-zearalenol (α-ZOL), which was up to 54 μg kg⁻¹ in coconut candy. As considers prevalence, aflatoxins and α-ZOL were not detected in lafun and groundnut-based snacks (donkwa and kulikuli), whereas each of the three mycotoxins contaminated 12.5% (1/8) of the coconut-based samples. This is the first report of α-ZOL in cassava and coconut, and their products. AFB₁ and total aflatoxins (TAFs) concentrations exceeded the maximum allowable limit recommended by National Agency for Food and Drug Administration and Control Nigeria (NAFDAC) in one sample of baked coconut (AFB₁ = 23 μg kg⁻¹ and TAFs = 26 μg kg⁻¹) and donkwa (AFB₁ = 19 μg kg⁻¹ and TAFs = 21 μg kg⁻¹).     

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.     

Determination of aflatoxins in walnut sujuk and Turkish delight by HPLC-FLD method

This study aims to assess the risk of aflatoxins (AFs) in traditional confectionery products (walnut sujuk and Turkish delight) of Turkey. A high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method was used for the determination of AFs. Evaluation of the method showed good selectivity, linearity, recovery and precision. The limit of quantification (LOQ) ranged from 0.106 to 0.374 μg kg⁻¹. The expanded measurement uncertainty was less than 40% for all target analytes. The validated method was successfully applied to the determination of AFs in 112 traditional confectionery products containing nuts (hazelnuts and walnuts). AFs were detected in 43.8% of walnuts and 60.9% of hazelnuts used as ingredients in walnut sujuk and Turkish delight and at levels ranging from 0.58 to 15.2 μg kg⁻¹ and 0.43–63.4 μg kg⁻¹, respectively. This means that AFs levels in walnut sujuk and Turkish delight were up to levels of 6.1 and 9.5 μg kg⁻¹, respectively. Six walnut samples and twenty-one hazelnut samples were above the EU maximum limits (MLs) of 2 and 5 μg kg⁻¹ for aflatoxin B₁ (AFB₁), respectively.     

A novel electrochemical piezoelectric label free immunosensor for aflatoxin B1 detection in groundnut

An electrochemical quartz crystal microbalance (EQCM) based label-free immunosensor has been developed for the quantitative detection of aflatoxin B1 (AFB1) in groundnut. The gold (Au) coated quartz crystal (6 MHz) functionalized with self-assembled monolayer (SAM) of 4-aminothiophenol (4-ATP) has been utilized to immobilize anti-aflatoxin antibodies (aAFB1). The quartz crystal microbalance QCM frequency and EQCM cyclic voltammetry measurements have been used to optimize the deposition of SAM layer on Au electrode (4-ATP/Au), antibody immobilization (aAFB1/4-ATP/Au), bovin serum albumin (BSA) adsorption (BSA/aAFB1/4-ATP/Au) and immuno interaction (AFB1/BSA/aAFB1/4-ATP/Au). Interestingly, the EQCM studies conducted on this immunoelectrode provide wider linear range of 0.1–4.0 ng mL⁻¹ and higher sensitivity than those obtained using under identical condition. The high association constant of 9.4 × 10⁻² ng mL⁻¹ of the EQCM immunosensor can be attributed to molecularly oriented self assembled monolayer of 4-ATP on QCM gold electrode that acts as a nanowire between aAFB1 and electrode through amide linkage formed by the NH₂ group of 4-ATP and COOH group of Fc part of aAFB1. This linkage also helps to project paratope part of aAFB1 to interact with AFB1. The developed label free EQCM based immunosensor was tested for the AFB1 detection in the extract of contaminated groundnut and the results were compared with Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The comparative results demonstrate the suitability of this sensor for routine screening of groundnut and the percent error is found to be ±12%.     

Aflatoxin M1 survey on randomly collected milk powder commercialized in Argentina and Brazil

Aflatoxin M₁ (AFM₁), the main monohydroxylated of aflatoxin B₁ (AFB₁) formed in liver and excreted in the milk, has toxicological proprieties comparable to those of AFB₁, albeit a lower carcinogenic potency. The presence of AFM₁ was investigated in 30 samples of powdered milk purchased in Argentina and Brazil. The samples were analyzed using an immunoaffinity column for cleanup and HPLC-FLD for determining AFM₁. The quantification limit was 0.1 μg/kg. AFM₁ was found in all the samples at levels ranging from 0.1 to 0.92 μg kg⁻¹ with average concentration of 0.39 μg kg⁻¹.     

Effect of water activity and temperature on the growth of Aspergillus flavus,the expression of aflatoxin biosynthetic genes and aflatoxin production in shelled peanuts

The contamination of peanuts with Aspergillus flavus and subsequent aflatoxins is considered to be one of the most serious safety problems in the world. Water activity (a_w) and temperature are limiting factors for fungal growth and aflatoxins production during storage. To optimize the practical storage parameter, the effect of a_w (0.85–0.99) and temperature (15–42 °C) on fungal growth, aflatoxin production and the expression of aflatoxin biosynthetic and regulatory genes in shelled peanuts was investigated. A. flavus grew at a lower rate when temperature ≤20 °C or a_w ≤ 0.85. For the growth of A. flavus in shelled peanuts, the optimum conditions were a_w was 0.98, and the optimum temperature was 37 °C. The maximum amount of AFB₁ in peanuts was obtained at 28 °C and a_w 0.96. Real-time analysis showed that 16 of 25 genes had highest expression levels at 28 °C under a_w 0.92, while 9 genes had highest expression levels at 37 °C under a_w 0.92. Compared with 37 °C, all aflatoxin biosynthetic pathway genes were down-regulated at 42 °C. All the pathway genes and laeA were up-expressed at a_w of 0.96 under 28 °C, compared to a_w 0.99. Furthermore, there was a good positive correlation between the ratio of aflS/aflR and AFB₁ production. The expression of laeA was also positively correlated with AFB₁ production while the expression of brlA was correlated with the A. flavus growth. The results of this study suggest that AFB₁ production in peanut kernels can occur over a wider range of a_w × temperatures levels compared to formula media and peanut media. Previous studies have showed that AFB₁ could not be produced on formula media at 37 °C without the expression of most aflatoxin structural genes. But, in the un-autoclaved shelled peanuts, high concentration of AFB₁ was produced at 37 °C with up-regulation of some aflatoxin biosynthetic genes. From a food safety point of view, the results can be used to optimize certain food technological processes and develop prevention strategies to control such carcinogenic natural metabolites in grains (such as peanuts, maize and rice) and derived products.     

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.     

Highly sensitive toxin microarray assay for aflatoxin B1 detection in cereals

We report a rapid, highly sensitive microarray method for quantitative aflatoxin B₁ (AFB₁) detection in cereal samples. Following optimisation using an indirect competitive immunoassay, optimised amounts of AFB₁-bovine serum albumin (AFB₁-BSA)-conjugate were contact-printed onto 16 isolated sub-arrays on multi-pad nitrocellulose coated slides subsequently used in competitive binding assays.The toxin microarray working range for AFB₁ was established in the range of 15 pg g⁻¹ to 3.04 ng g⁻¹, with a detection limit of 1 pg g⁻¹. To determine assay sensitivity in contaminated food models, wheat flour and barley grains samples were spiked with AFB₁ standard dilutions. Following extraction, the working ranges of 0.11–4.15 and 0.18–4.31 ng g⁻¹ were determined, with detection limits of 30 and 90 pg g⁻¹, respectively. The sensitivity of the developed assay is below the European commission limit set for AFB₁ detection and the assay procedure was completed in 3 h time. Good recoveries (98% ± 11%) obtained demonstrate the suitability of the proposed method for rapid and sensitive quantification of AFB₁ in contaminated cereal samples.