Aflatoxin-producing potential of Aspergillus flavus and Aspergillus parasiticus isolated from samples of smoked-dried meat

Over a period of three years 420 samples of various smoke-dried meat products, collected from individual households in different region of Croatia were analysed for the presence of aflatoxigenic strains of the Aspergillus flavus group. Strains of A. flavus and A. parasiticus were present in 17,8% of the samples, and aflatoxin-producing ability was tested in 75 strains. In relation to sequential method of aflatoxin detection, 5 of 8 isolates were found in the first step (fluorescence in aflatoxin-producing ability medium - APA) and all of them in the second step (extraction method from syntheses on moist shredded wheat - SW). A. flavus strains produced mainly aflatoxin B₁, and had various levels of toxigenicity (1.4–3.12 mg/kg). Some strains of A. parasiticus produced all four aflatoxins B₁ B₂ G₁ G₂, while the other ones produced AF B₁ + G₁ only, with concentrations of aflatoxins from 0.1 to 450 mg/kg.     

Isolation, enumeration and PCR characterization of aflatoxigenic fungi from food and feed samples in India

A total of 54 market samples comprising nine different food and feed commodities from Mysore city were examined in order to isolate aflatoxin-producing fungi as well as to assess aflatoxins in the commodities. Thirty-two samples were contaminated with aflatoxigenic fungi and the total mycoflora and aflatoxigenic fungi in different food and feed commodities were in the range of 0.2–260 and 0–100 cfu×10³/g, respectively. In total, 136 fungi were isolated, of which 32 were Aspergillus flavus strains and 26 of them were found to produce aflatoxins. A. flavus group of fungi comprising A. flavus, A. parasiticus, A. oryzae, A. sojae were characterized by using Aspergillus differential medium and PCR. The PCR was performed using two different sets of primers specifically targeted to aflR and omt genes of aflatoxin biosynthesis pathway. Most of the fungi belonging to A. flavus group reacted positively with the primers resulting in expected size amplicons of 796 bp for aflR and 404 bp for omt. Among the nine commodities screened for aflatoxin only, groundnut and groundnut cake were contaminated with aflatoxins B₁ and B₂. The aflatoxin contamination in these commodities exceeded the Indian regulatory limit of 30 μg/kg.     

Aflatoxins and heavy metals in animal feed in Iran

The occurrence of aflatoxin (aflatoxin B₁, aflatoxin B₂, aflatoxin G₁ (AFG₁) and aflatoxin G₂ (AFG₂)) and heavy metal (Pb, Cd, As and Hg) contamination was determined in 40 industrially produced animal feed samples which were collected from the southwest of Iran. The results indicated that 75% of samples were contaminated by four aflatoxins and the level of AFB₁ and sum of aflatoxins were higher than the permissible maximum levels in Iran (5 and 20 µg kg^?1, respectively) in all feed samples. A positive correlation was found between four types of aflatoxins in all the tested samples (p < 0.01) and the positive correlation between AFG₁ and AFG₂ was significant (r² = 0.708). All feed samples had lead concentrations lower than the maximum EU limit, while 5%, 17% and 42.5% of feed samples had As, Cd and Hg concentrations higher than the maximum limits, respectively.     

Moulds and mycotoxins in rice from the Swedish retail market

A survey of moulds and mycotoxins was performed on 99 rice samples taken from the Swedish retail market. The main objective was to study the mould and mycotoxin content in basmati rice and rice with a high content of fibre. Samples of jasmine rice as well as long-grain rice were also included. The samples were analysed for their content of ochratoxin A (high-performance liquid chromatography (HPLC)), aflatoxin B₁, B₂, G₁, and G₂ (HPLC, RIDA®QUICK), and mould (traditional cultivation methods in combination with morphological analysis). The majority of samples were sampled according to European Commission Regulation 401/2006. Subsamples were pooled and mixed before milling and both mould and mycotoxin analyses were performed on milled rice. The results showed that the majority of basmati rice (71%) and many jasmine rice samples (20%) contained detectable levels of aflatoxin B₁ (level of quantification = 0.1 µg aflatoxin kg^?1 rice). Two samples of jasmine rice and ten basmati rice samples contained levels over the regulated European maximum limits of 2 µg kg^?1 for aflatoxin B₁ or 4 µg kg^?1 for total aflatoxins. Aspergillus was the most common mould genus isolated, but also Penicillium, Eurotium, Wallemia, Cladosporium, Epicoccum, Alternaria, and Trichotecium were found. The presence of Aspergillus flavus in 21% of the samples indicates that incorrect management of rice during production and storage implies a risk of mould growth and subsequent production of aflatoxin. Rough estimates showed that high rice consumers may have an intake of 2–3 ng aflatoxin kg^?1 bodyweight and day^?1 from rice alone. This survey shows that aflatoxin is a common contaminant in rice imported to Europe.     

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.     

Aspergillus and aflatoxin in groundnut (Arachis hypogaea L.) and groundnut cake in Eastern Ethiopia

This study was conducted to assess major Aspergillus species and aflatoxins associated with groundnut seeds and cake in Eastern Ethiopia and evaluate growers’ management practices. A total of 160 groundnut seed samples from farmers’ stores and 50 groundnut cake samples from cafe and restaurants were collected. Fungal isolation was done from groundnut seed samples. Aspergillus flavus was the dominant species followed by Aspergillus parasiticus. Aflatoxin analyses of groundnut seed samples were performed using ultra performance liquid chromatography; 22.5% and 41.3% of samples were positive, with total aflatoxin concentrations of 786 and 3135 ng g^?1 from 2013/2014 and 2014/2015 samples, respectively. The level of specific aflatoxin concentration varied between 0.1 and 2526 ng g^?1 for B₂ and B₁, respectively. Among contaminated samples of groundnut cake, 68% exhibited aflatoxin concentration below 20 ng g^?1, while as high as 158 ng g^?1 aflatoxin B₁ was recorded. The study confirms high contamination of groundnut products in East Ethiopia.     

Spoilage moulds and aflatoxins from poultry feeds

Aflatoxin producing strains of Aspergillus flayus Link (IMI 280819) and A. oryzae (Ahlb.) Cohn (IMI 280831) were among the eleven spoilage moulds isolated from five types of poultry feeds. The recorded pH and moisture content values of the various feeds are conducive to mould deterioration. All the four principal aflatoxins (B₁, B₂, G₁, G₂) were detected in the analysed feeds though at toxicologically ‘safe’ levels for most farm animals. Significant quantities of aflatoxin B₁ were produced by the two fungal isolates in all the five classes of poultry feeds with A. flavus yielding the larger amounts. Optimum aflatoxin B₁ production and mycelial growth in chick mash infusion medium were recorded for both species at 30 and 35 °C, respectively and similarly on the 8th and 6th day respectively when cultures were incubated at 30 °C.     

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.     

Mycotoxic flora and mycotoxins in smoke-dried fish from Sierra Leone

Examination of 20 samples of smoke-dried fish of the Ethmolosa sp. commonly called “Bonga”, from homes and markets in Njala (Sierra Leone) revealed the presence of 4 Aspergilli species: A. flavus Links ex Fries, A. ochraceus Wilhelm, A. tamarii Kita and A. niger van Tieghem. Fresh or properly preserved smoke-dried fish showed no signs of fungal contamination. Mouldy fish extracts contained varying amounts of aflatoxins B₁, G₁, G₂ and ochratoxin A. Isolates of A. flavus grown on yeast extract sucrose (YES) medium, produced considerable amounts of aflatoxin B₁ and G₁ and trace amounts of G₂. On YES medium A. ochraceus produced large amounts of ochratoxin A but no penicillic acid.     

Mycotoxin contamination of sorghum and its contribution to human dietary exposure in four sub-Saharan countries

This research aimed at evaluating the safety, and the type, level and prevalence of mycotoxins in grain sorghum of four sub-Saharan African (SSA) countries (Burkina Faso, Ethiopia, Mali and Sudan). A multi-analyte LC-MS/MS method for quantification of 23 mycotoxins (nivalenol, deoxynivalenol, fusarenon X, neosolaniol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, diacetoxyscirpenol, roquefortine C, HT-2 toxin, alternariol, T-2 toxin, FB1, FB2, FB3, zearalenone, aflatoxin G₁, aflatoxin G₂, aflatoxin B₁, aflatoxin B₂, sterigmatocystin, OTA, altenuene, alternariol monomethylether) was applied to different sorghum matrices. Of the 1533 analysed samples, 33% were contaminated with at least one of the following mycotoxins: aflatoxins, fumonisins, sterigmatocystin, Alternaria toxins, OTA and zearalenone. Country of origin, colour, source and collection period of sorghum samples significantly influenced the type, level and prevalence of mycotoxins. Sterigmatocystin (15%), fumonisins (17%) and aflatoxins (13%) were the most prevalent. FB1 (274 ± 585 µg/kg) had the highest mean concentration followed by FB2 (214 ± 308 µg/kg) while diacetoxyscirpenol (8.12 ± 19.2 µg/kg) and HT-2 (11.9 ± 0.00 µg/kg) had the lowest concentrations. Neosolaniol, fusarenon-X, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, T-2 toxin, nivalenol and roquefortine C were not detected in any of the samples. Sudan had the lowest prevalence and mean concentration of all mycotoxins. Pink sorghum had the highest concentrations of fumonisins and aflatoxins. Mycotoxins from Aspergillus spp. and Alternaria spp. are the mycotoxins of concern in SSA grain sorghum with regard to prevalence, concentration and possible health risk from exposure. Based on the performed risk characterisation, daily consumption of sorghum containing aflatoxins, alternariol, alternariol monomethyl ether, sterigmatocystin and OTA could result in exceeding the established health-based guidance values for these toxins.     

Effect of synthetic antioxidants on stored maize grain mycoflora in situ

BACKGROUND: The influence of a mixture of butylated hydroxyanisole (BHA) and propyl paraben (PP) (each at a concentration of 20 mmol L^?1) on mycoflora and Aspergillus section Flavi populations in stored maize grain was evaluated. A survey of 120 maize samples was carried out from June to November 2005. RESULTS: The predominant populations in non‐treated (control) maize between the first and sixth sampling periods were Aspergillus section Flavi and Penicillium. Aspergillus flavus was the fungus most frequently isolated from both control and antioxidant‐treated kernels. All samples of control and antioxidant‐treated maize kernels were negative for aflatoxins during the 6 month storage period. Aspergillus flavus and A. parasiticus strains showed a variable ability to produce aflatoxins. The contribution of the strains to silo community toxigenicity was higher for A. flavus L (large) and S (small) strains in the fourth sampling period. CONCLUSION: Antioxidant treatment negatively affected natural maize mycoflora and Aspergillus section Flavi populations between the second and sixth months of storage. Copyright © 2007 Society of Chemical Industry     

Water availability and calcium propionate affect fungal population and aflatoxins production in broiler finisher feed during storage

The aim of this study was to investigate the effects of calcium propionate, water activity (a_w) and incubation time on the total fungal count and aflatoxins B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂) production in the broiler finisher feed. The feed was added with calcium propionate (5 g kg^–1), adjusted to 0.85, 0.90 and 0.95 a_w and stored for 28 days at 25°C, analysing for mould growth and aflatoxins production every 7 days. Analysis of variance indicated that all the factors (preservative, a_w and storage time) alone and in combination significantly (p < 0.001) affected the total fungal count and aflatoxins production in the feed. Minimum total fungal counts (1.99 × 10² CFU g^–1) were observed in calcium propionate feed at 0.85 a_w on day 1 and the highest (4.36 × 10⁹ CFUs g^–1) in control sample at 0.95 a_w on day 28 of storage. During the storage period, AFB₁ content in control samples increased from 11.35 to 73.44, from 11.58 to 81.81 and from 11.54 to 102.68 ng g^–1, whereas in preserved feed the content of B₁ increased from 11.47 to 37.83, from 11.54 to 49.07 and from 11.20 to 53.14 ng g^–1 at 0.85, 0.90 and 0.95 a_w, respectively. Similar patterns were noted for AFB₂, AFG₁ and AFG₂ contents. All the aflatoxins readily increased over storage time; however, the increase was much slower in preserved feed that contained a lower amount of available water. This study reveals that calcium propionate addition to poultry litter along with water activity amelioration is an effective tool for controlling mould incidence and aflatoxin production in poultry feed.     

Chemical composition of Ocimum basilicum L. essential oil and its efficacy as a preservative against fungal and aflatoxin contamination of dry fruits

The study presents fungal and aflatoxin contamination of some dry fruits and Ocimum basilicum essential oil (EO) as a plant‐based preservative. During mycoflora analysis, 2045 fungal isolates were recorded from dry fruits and 40% isolates of Aspergillus flavus were toxigenic in nature. The EO of O. basilicum exhibited strong fungitoxicity against toxigenic strain of A. flavus. Its minimum inhibitory concentration (MIC) was recorded at 1.0 μL ml^?1, and it completely inhibited aflatoxin B₁ production at 0.5 μL ml^?1. The oil exhibited broad fungitoxic spectrum and considerably reduced A. flavus isolates from dry fruits when used as fumigant in closed storage containers at 1.0 μL ml^?1. The chemical profile of the EO was standardised through GC–MS analysis. Based on antifungal potency, antiaflatoxigenicity and efficacy as fumigant during storage conditions, O. basilicum EO may be recommended as a botanical preservative for enhancing the shelf life of dry fruits and edible products during storage.     

Aflatoxins,ochratoxin A and zearalenone in Brazilian corn cultivars

Past surveys indicated that the occurrence of aflatoxins, zearalenone and ochratoxin A was not a problem in corn and corn products in the state of São Paulo, Brazil. However, according to recent studies, a change in pattern has been detected. To obtain a better overview, these toxins were searched for in 110 samples of freshly harvested corn, corresponding to 48 commercial cultivars planted at three different locations in the state. Aflatoxin contamination was found in 60 (54.5%) of the samples, in levels ranging from 6 to 1600 µg kg^?1 aflatoxin B₁. Insect control was exercised, so this was not the main route of corn infection. Endosperm type, germplasm type, number of days to flowering, and length of time the mature corn remained in the field had no effect on aflatoxin contamination. Ochratoxin A was found in two samples (206 and 128 µg kg^?1) and zearalenone in one sample (4640 µg kg^?1). Possible causes of the increase in aflatoxin levels may lie in the changing nature of the commercial cultivars employed, associated with the forsaking of the original landraces, and in a change in the toxigenicity pattern of the corn mycoflora Aspergillus flavus/Aspergillus parasiticus prevailing strains. © 2001 Society of Chemical Industry     

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.     

The effect of village processing techniques on the content of aflatoxins in corn and peanuts in Zambia

The effect of village processing techniques on the aflatoxin content of corn and peanut products was investigated. In 30 trials, corn kernels were dehulled (bran removal), soaked for 24 h, washed and dried before grinding into flour and boiling in water to a thick consistency (Nshima). Shelled peanuts were either dry-roasted as whole kernels or ground into peanut meal and cooked. Dehulling, following by 24-h soaking (steeping) and subsequent washing significantly (P<0·05) reduced the aflatoxin B₁ content of corn flour from 900 to 150 μg kg⁻¹, and similarly that of aflatoxin G₁ from 929 to 114 μg kg⁻¹. Preparation of Nshima did not result into a substantial reduction in aflatoxin content, neither did extension of the cooking duration of 2 h afford any further decontamination. Whereas boiling peanut meal yielded a moderate reduction in the content of aflatoxins B₁ and G₁, roasting whole peanut kernels greatly reduced (P<0·001) the concentrations of the toxins from that in raw kernels (AFB₁= 8600 μg kg⁻¹ and AFG₁=6200 μg kg⁻¹) to 1300 and 1200 μg kg⁻¹, respectively. These results indicate that specific processing techniques carried out in Zambian villages are effective in reducing aflatoxin carry-over into edible fractions, while others are not. © 1998 SCI.     

Intake of aflatoxins through the consumption of peanut products in Brazil

To estimate daily intake of aflatoxins from peanut products consumed by the population of Paraná State (Brazil), 100 samples of peanut products were collected between July 2006 and April 2007. Aflatoxins were determined by an HPLC method with fluorescence detection. There was a 50% occurrence of aflatoxins (B₁, B₂, G₁ and G₂) in concentrations ranging from 0.5 to 113?ng?g^?1, with 13 samples with levels above 20?ng?g^?1. Intake was calculated for average and high adult consumers of peanut products and it was compared with provisional maximum tolerable daily intake (PMTDI). The estimated probable daily intake (PDI) for aflatoxin B₁ (AFB₁) varied from 0.6 to 10.4?ng?kg^?1?bw?day^?1, exceeding the PMTDI of 0.4?ng?kg^?1?bw?day^?1 for carriers of hepatitis B virus.     

Modellversuche zur Aflatoxinbildung in Schmelzk?se

Zusammenfassung Schmelzkäse stellt fürAspergillus favus ein sehr gutes Substrat dar. Nach 16 tägiger Kulturdauer konnten folgende Aflatoxinmengen nachgewiesen werden: 35 ppm Aflatoxin B₁, 66 ppm Aflatoxin G₁, 8,6 ppm Aflatoxin B₂, 5,3 ppm Aflatoxin G₂ und 2,1 ppm Aflatoxin M₁. Eine Erhöhung der Schmelzsalzkonzentration von 3% auf 8% bzw. ein Zusatz von 6% Natriumchlorid reduzierten das Toxinbildungsvermögen vonAspergillus favus deutlich.

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Simulation of natural mould growth in processed cheese

Summary Processed cheese is a very good substrate forAspergillus flavus. After 16 days incubation, a processed cheese inoculated withAspergillus flavus was found to contain 35 ppm aflatoxin B₁, 66 ppm aflatoxin G₁, 8.6 ppm aflatoxin B₂, 5.3 ppm aflatoxin G₂ and 2.1 ppm aflatoxin M₁. An increase in the content of emulsifying salt from 3% to 8% caused a distinct decrease in the contents of aflatoxin B₁ and G₁ in the cheese, as did the addition of 6% sodium chloride.

     

Occurrence of fumonisins and aflatoxins in cereals from markets of Hebei province of China

Fumonisins B₁, B₂ and B₃ (FB₁, FB₂ and FB₃) and aflatoxins B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂) are both major mycotoxins of food concern, because of their wide range of concentration and possible co-occurrence. Therefore, a contamination survey in corn and wheat flour by liquid chromatography–tandem mass spectrometry was carried out. Quantification of fumonisins and aflatoxins was based on internal calibration (by the use of ¹³C₃₄-fumonisin) and external calibration, respectively. Fumonisins were detected in 95% of corn samples and in 7% of wheat flour samples, with the mean level (FB₁?+?FB₂?+?FB₃) of 441?µg?kg^?1 and 0.09?µg?kg^?1, respectively. Low levels of aflatoxins were detected in 37% of the samples with a mean level (B₁?+?B₂?+?G₁?+?G₂) of 0.12?µg?kg^?1. Fumonisins and aflatoxins were not detected in 29% of the samples analysed. Simultaneous occurrence of fumonisins and aflatoxins was observed in 12% of samples.     

Fungi associated with post-harvest rot of sweet orange (Citrus sinensis) and aflatoxin B1 production by isolates of Aspergillus flavus on plain and supplemented orange juice

Samples of rotting sweet orange (Citrus sinensis) were obtained from the depots, sales counters and waste baskets. Fungi associated with rotting fruits were isolated and identified. Out of 12 species of fungi isolated, 8 are known to be producers of toxins. The 7 isolates of Aspergillus flavus obtained were screened for aflatoxin production in a nutrient solution, and 4 were found to be aflatoxigenic, producing primarily aflatoxin B₁. Aflatoxin B₁ production of the toxigenic isolates were further studied on plain juice and juice separately supplemented with 2.0% yeast extract and 2.0% sucrose. The highest yield of aflatoxin B₁ was produced on juice supplemented with yeast extract by the 4 toxigenic A. flavus isolates, followed by sucrose supplementation while the lowest amount of aflatoxin B₁ was detected on plain juice. Optimum temperature for aflatoxin B₁ production by A. flavus isolate (IBA-O1) was 25 °C to 30 °C, for an incubation period of 7–11 days on plain and supplemented juice media.