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.     

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.     

Aflatoxigenic fungi and aflatoxin in cocoa

This paper reports the occurrence of aflatoxigenic fungi and the presence of aflatoxins in 226 cocoa samples collected on Brazilian farms. The samples were taken at various stages of fermentation, drying and storage. A total of 819 potentially aflatoxigenic fungi were isolated using Dichloran 18% Glycerol agar after surface disinfection, and identified by standard techniques. The ability of the fungi to produce aflatoxins was determined using the agar plug technique and TLC. The presence of aflatoxins in cocoa samples was determined by HPLC using post-column derivatization with bromide after immunoaffinity column clean up. The aflatoxigenic fungi isolated were Aspergillus flavus, A. parasiticus and A. nomius. A considerable increase in numbers of these species was observed during drying and storage. In spite of the high prevalence of aflatoxigenic fungi, only low levels of aflatoxin were found in the cocoa samples, suggesting the existence of limiting factors to the accumulation of aflatoxins in the beans.     

Occurrence of aflatoxins in milk thistle herbal supplements

Milk thistle (MT) dietary supplements are widely consumed due to their possible liver-health-promoting properties. As botanicals they can be contaminated with a variety of fungi and their secondary metabolites, mycotoxins. The aflatoxigenic fungus Aspergillus flavus has been previously isolated from these commodities. Currently, there is no published method for determining aflatoxins (AFs) in MT. Therefore, a liquid chromatography (LC) method validated for aflatoxin analysis in botanicals was evaluated and applied to MT. The method consisted of acetonitrile/water extraction, immunoaffinity column clean-up, LC separation, post-column photochemical reaction derivatisation and fluorescence detection. The average recoveries for AFs added to MT seeds, herb, oil-based liquid extract and alcohol-based liquid extract were 76% or higher. The mean relative standard deviation was <10%. The limit of detection (LOD) was 0.01?µg kg^?1 and the limit of quantification (LOQ) was 0.03?µg kg^–1. The method was used to conduct a small survey. A total of 83 MT samples from the US market were analysed. AFs were detected in 19% of the samples with levels ranging from 0.04 to 2.0?µg kg^–1. Additionally, an aflatoxigenic A. flavus strain from ATTC and an A. parasiticus strain isolated from MT herb powder were found to produce high amounts of aflatoxins (11,200 and 49,100?µg kg^–1, respectively) when cultured in MT seed powder. This is the first study reporting on aflatoxin contamination of MT botanical supplements and identifying methodology for AF analysis of these commodities.     

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.     

Multiplex PCR assay for the detection of aflatoxigenic and non-aflatoxigenic fungi in meju, a Korean fermented soybean food starter

Aflatoxins are toxic secondary metabolites produced commonly by Aspergillus flavus and Aspergillus parasiticus. In this study, the possibility of using multiplex PCR was investigated to speed up and specify the detection of aflatoxigenic Aspergillus species in meju, a traditional Korean fermented soybean food starter. Two different sets of three primers were designed specifically for the omtB, ver-1, aflR, and omtA genes present in the aflatoxin biosynthesis cluster. The optimized multiplex PCR showed that only aflatoxigenic Aspergillus species gave three band patterns in both primer sets. The detection limits were determined as 125 pg/μl for genomic DNA from aflatoxigenic A. parasiticus KCCM 35078, and 10⁵ spores/g of meju sample for DNA extracted directly from meju. A total of 65 Aspergillus isolates from meju were tested for the presence of aflatoxigenic fungi by the application of multiplex PCR, and were analyzed by TLC and HPLC for the aflatoxin production in the culture filtrates. Results showed a good correlation between the presence of the aflatoxin biosynthesis genes analyzed by multiplex PCR and aflatoxin production by TLC and HPLC. This suggests that this multiplex PCR method may provide an accurate and specific detection of aflatoxigenic Aspergillus species in fermented soybean foods.     

The production of aflatoxins in fresh date fruits and under simulated storage conditions

Sixteen varieties of date fruit (Phoenix dactylifera) at three stages of maturation (Kimri, Rutab and Tamr) were examined for the presence of fungi and analysed for aflatoxins B₁, B₂, G₁ and G₂ and sterigmatocystin. Single samples of each variety were used in the study. Samples as received were initially examined for mycoflora and toxin levels and then stored at 98% relative humidity and 30 °C for 14 days to investigate the effects of possible adverse storage conditions on mycoflora and, in particular, aflatoxin formation. All samples showed an absence of aflatoxins and their precusor, sterigmatocystin, after adverse storage for 14 days, although aflatoxin‐producing Aspergillus flavus isolates were identified in 10 varieties at the first stage of maturation (Kimri). High fungal counts were associated with the Rutab stage and low counts with the Tamr stage. The counts of A flavus ranged from 5.00 to 8.16 log₁₀(cfu g^?1) under simulated storage conditions, and three varieties contained significant levels of aflatoxin B₁ or B₂ ranging from 35 to 11 610 µg kg^?1. Sterigmatocystin was not detected in any of the samples as received or under simulated storage conditions. © 2002 Society of Chemical Industry     

Assessment of Thymus vulgaris L. essential oil as a safe botanical preservative against post harvest fungal infestation of food commodities

A total of 14 odoriferous angiospermic essential oils were tested against the toxigenic strain of Aspergillus flavus. The essential oil of Thymus vulgaris L. showed highest antifungal efficacy. The thyme oil absolutely inhibited the mycelial growth of A. flavus at 0.7 μl ml^− 1 and exhibited a broad fungitoxic spectrum against eight different food contaminating fungi viz. Fusarium oxysporum, Cladosporium herbarum, Curvularia lunata, Aspergillus terreus, Aspergillus niger, Aspergillus fumigatus, Alternaria alternata and Botryodiploidia theobromae. The oil also showed significant antiaflatoxigenic efficacy as it completely arrested the aflatoxin B₁ production at 0.6 μl ml^− 1. Thyme oil as fungitoxicant was also found superior over most of the prevalent synthetic fungicides. The LC₅₀ of thyme oil against mice was recorded as 7142.85 μl kg^− 1 body weight indicating its non-mammalian toxicity and strengthening its safe exploitation as preservative for stored food commodities. The findings recommend the thyme oil as potential botanical preservative in eco-friendly control of biodeterioration of food commodities during storage.

Industrial relevance

The thyme essential oil may be recommended for large scale application as a plant based preservative for stored food items because of its strong antifungal as well as antiaflatoxigenic efficacy. Because of broad antimicrobial spectrum, more efficacy over prevalent synthetic preservatives as well as non-mammalian toxicity, the thyme essential oil may be formulated as a safe and economical plant based preservative against post harvest fungal infestation and aflatoxin contamination of food commodities.     

Assessment of Pelargonium graveolens oil as plant‐based antimicrobial and aflatoxin suppressor in food preservation

BACKGROUND: Contamination of stored food commodities by moulds and mycotoxins results in qualitative as well as quantitative losses. Most of the synthetic antimicrobials used for preservation of stored food items produce side effects in the form of residual and mammalian toxicity. Recently some higher plant products have been recommended as safe alternatives of such synthetic antimicrobials. In the present investigation antifungal efficacy of some essential oils was evaluated against two toxigenic strains of Aspergillus flavus with special reference to the oil of Pelargonium graveolens to investigate its potential to inhibit aflatoxin B₁ secretion. RESULTS: Essential oil of P. graveolens exhibited absolute fungitoxicity against both the toxigenic strains of A. flavus. The minimum inhibitory concentration of the oil was found to be 0.75 g L^?1 and exhibited a fungistatic nature. It was found superior over the synthetic fungicides tested and exhibited a broad fungitoxic spectrum. The oil showed excellent anti‐aflatoxigenic efficacy as it completely inhibited aflatoxin B₁ production even at 0.50 g L^?1. CONCLUSION: This is the first report on the aflatoxin B₁ inhibitory nature of P. graveolens oil. It may be recommended as a novel plant‐based antimicrobial as well as aflatoxin B₁ suppressor over synthetic preservatives in food protection. Copyright © 2008 Society of Chemical Industry     

Aflatoxins distribution in fractions derived from tofu production

ABSTRACT

Tofu or bean curd is obtained from soybean seeds being a widespread food product in Asia. The commodity used for its production can be contaminated with aflatoxins, which are secondary metabolites synthetised by species of Aspergillus flavus and A. parasiticus. Intake of contaminated food products causes toxic effects on consumers. The aim of this work was to study aflatoxin distribution in fractions obtained from pilot-scale tofu production with contaminated soybeans. The presence of the aflatoxins B₁, B₂, G₁ and G₂ (AFs) in soaking water, okara, whey and tofu was analysed. Aflatoxin analysis was performed by HPLC with fluorescence detection. The distribution of aflatoxins in all the analysed fractions was not a normal distribution. The liquid fractions (soaking water and whey) had less contamination than solid fractions (tofu and okara). The percentage AFB₁ remaining in nutritionally important fractions, okara and tofu, was between 6.2% and 67.7% (median = 18.1%) and 0.5% and 13.2% (median = 3.5%), respectively. AFB₂, AFG₁ and AFG₂ had a similar distribution. These results showed that throughout tofu production, AFs can be present in the products intended for human consumption.     

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 growth and aflatoxin production of Aspergillus flavus strains on a cheese model system are influenced by physicochemical factors

Traditional cheeses may be contaminated by aflatoxin-producing Aspergillus flavus during the ripening process, which has not been sufficiently taken into account. The objectives of this study were to evaluate the influence of water activity (a_w), pH, and temperature on the lag phases, growth, and aflatoxin production of 3 A. flavus strains (CQ7, CQ8, and CG103) on a cheese-based medium. The results showed that the behavior of A. flavus strains was influenced by pH, a_w, and temperature conditions. The CQ7 strain showed the maximum growth at pH 5.5, 0.99 a_w, and 25°C, whereas for CQ8 and CQ103 strains, no differences were obtained at pH 5.5 and 6.0. In general, low pH, a_w, and temperature values increased the latency times and decreased the growth rate and colony diameter, although a_w and temperature were the most limiting factors. Maximum aflatoxin production on the cheese-based medium occurred at pH 5.0, 0.95 a_w, and 25 or 30°C, depending on the strain. This study shows the effect of pH, a_w, and temperature factors on growth and aflatoxin production of 3 aflatoxigenic A. flavus strains on a cheese-based medium. The findings may help to design control strategies during the cheesemaking process and storage, to prevent and avoid aflatoxin contamination by aflatoxigenic molds.     

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.     

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.     

Application of hazard analysis critical control points system for the control of aflatoxins in the Brazilian groundnut‐based food industry

This study was conducted to identify and assess the critical control points (CCPs) in groundnut‐based food production in southern Brazil in order to reduce or eliminate aflatoxin contamination. This methodology has been suggested by the Food and Agriculture Organization (FAO) of the United Nations. Reception of prime matter, groundnut storage, roasting and thermal treatment were the main CCPs identified. Critical factors were the determination of aflatoxin, moisture content and water activity (A_w) during groundnut reception and storage, control of temperature, roasting time and thermal treatment in the groundnut‐based food manufacturing. The critical limit for moisture was 8.2% and 0.6 was established for A_w. In Brazil, the limit for aflatoxins B₁, B₂, G₁ and G₂ has been established at 20 ppb. Temperatures of 180 °C/1 h and 80 °C/40 min were established for roasting and thermal treatment stages of groundnut‐based food, respectively.     

Efficacy of bioactive compounds isolated from Albizia amara and Albizia saman as source of antifungal and antiaflatoxigenic agents

The antifungal and antiaflatoxigenic activities of budmunchiamine A (BUA) isolated from Albizia amara and pithecolobine (PI) isolated from Albizia saman were assessed. The present study reports the broad-spectrum and concentration-dependent antifungal activities of BUA and PI with zone of inhibitions and minimum inhibitory concentrations ranging from 6.8 to 19.6 mm and 0.015–0.5 mg/mL, respectively. Aspergillus flavus growth and its aflatoxin B₁ production were completely inhibited in vitro by BUA and PI at concentration of 1 mg/mL. BUA severely inhibited the growth of wide range of seed-borne fungi of maize including aflatoxigenic A. flavus with an increased seedling vigour index in vivo at a lower concentration (1.0 g/kg) than PI (2.0 g/kg). Enhanced seedling vigour was observed in BUA and PI treatments with no adverse effect on seed germination. The present findings indicate the possible use of BUA and PI as antifungal agents against post harvest fungal infestation of food commodities and mycotoxin contamination.     

Contamination of freshly harvested Bambara groundnut (Vigna subterranea) seed from Mpumalanga,South Africa,with mycotoxigenic fungi

Freshly harvested Bambara groundnut (BGN) is occasionally consumed raw and can potentially become infected with mycotoxingenic field fungi. In this study, BGN samples were obtained from 12 farms in three districts of Mpumalanga in South Africa. Eight pooled samples were screened for multi-mycotoxin contamination using Ultra Performance Liquid-Chromatography tandem mass spectrometry (UPLC-MS/MS). To identify mycoflora, 12 samples were screened using conventional and molecular methods. Selected potential mycotoxin producing isolates were screened for mycotoxins using UPLC-MS/MS. No mycotoxins were detected on the freshly harvested BGN samples, but they were infected with various mycotoxin producing fungal species namely Aspergillus flavus (50%), Penicillium citrinum (25%), Penicillium oxalicum (17%), Penicillium citreoviridin (0.8%), and Fusarium verticillioides (0.8%). Following screening of selected fungal cultures, aflatoxin B₁ (0.4, 0.45 and 0.4 ppm) and fumonisin B₁ (0.7 ppm) were detected from A. flavus and F. verticillioides, respectively. Identification of mycotoxigenic fungi on freshly harvested BGN presents a potential health risk.     

Antiaflatoxigenic and antioxidant activity of an essential oil from Ageratum conyzoides L.

BACKGROUND: Aflatoxin contamination of various commodities can occur as a result of infection, mainly by Aspergillus flavus and Aspergillus parasiticus. Every year, almost 25% of the world's food supply is contaminated by mycotoxins. Aflatoxins B₁, B₂, G₁ and G₂, which occur naturally, are significant contaminants of a wide variety of commodities. A number of biological activities have been associated with Ageratum conyzoides. We have therefore investigated the antiaflatoxigenic, antioxidant and antimicrobial activity of essential oils of A. conyzoides. This could help to turn A. conyzoides, a nuisance weed, into a resource. RESULTS: The essential oil of Ageratum conyzoides L. shows the presence of 12 compounds when analyzed by gas chromatography–mass spectrometry. The growth and aflatoxin production of the toxigenic strain Aspergillus parasiticus was completely inhibited by essential oil. All the studied concentrations of the oil demonstrate a reduction in mycelia growth and decreased production of different aflatoxins in fungi, as revealed by liquid chomatographic–tandem mass spectrometric analysis. Volatiles from macerated green leaf tissue of A. conyzoides were also effective against A. parasiticus. The strongest antibacterial activity was observed against the bacteria Staphylococcus aureus and Bacillus subtilis in a disk diffusion bioassay. Essential oil and methanol extract of A. conyzoides L. were assayed for their antioxidant activity. Methanol extract showed the highest antioxidant activity in FRAP and DPPH assay, whereas essential oil showed greater lipid peroxidation inhibition than methanol extract. CONCLUSION: The plant's ethno‐medicinal importance, antioxidant potential, inhibitory activity against the Aspergillus group of fungi and production of aflatoxins may add a new dimension to its usefulness in the protection of stored product. Copyright © 2010 Society of Chemical Industry     

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.