Detection of moulds producing aflatoxins in maize and peanuts by an immunoassay

An enzyme-linked immunosorbent assay (ELISA) was developed to detect moulds producing aflatoxins in maize and peanuts by an antibody produced to extracellular antigen from Aspergillus parasiticus. This antibody recognized species with phenotypic similarities to A. parasiticus, A. flavus and the domesticated species A. sojae and A. oryzae. For maize samples that were naturally contaminated with aflatoxins, low and high levels of aflatoxin corresponded with low and high ELISA readings for mould antigens, respectively. Maize and peanuts inoculated with 10(2) spores ml(-1) of A. parasiticus and incubated at 15 degrees C for 18 days or 21 degrees C for 7 days were analyzed for mould antigens and aflatoxin levels. At 15 degrees C, mould antigens were detected by day 4 in maize when 0.16 ng g(-1) of aflatoxin was detected by ELISA but not by thin layer chromatography (TLC). Antigens were detected in peanuts by day 4 before aflatoxin was found. Likewise, at 21 degrees C, antigens were detected by day 4 in maize when less than 1 ng g(-1) of aflatoxin was detected by ELISA but not by TLC, but by day 2 in peanuts when no aflatoxin was detected. A. parasiticus could be detected before it could produce aflatoxins. Therefore, this ELISA shows potential as an early detection method for moulds that produce aflatoxins.     

Mycoflora of hazelnut (Corylus avellana L.) and aflatoxin content in hazelnut kernels artificially infected with Aspergillus parasiticus

In this research, 30 hazelnut samples were used to determine their internal mould populations and their aflatoxin contents. The results showed that Aspergillus was the most common genus in the experimental samples (96.6% of all samples). Thirty-one% of isolates were classified as Aspergillus flavus and none of the samples contained aflatoxins. In the second part of the experiment, the kernels were inoculated with conidia of Aspergillus parasiticus and incubated at three different humidified conditions and two different temperatures for 45 days. Aflatoxin contents of the samples kept in 98% relative humidity and at 28 degrees C were higher (904.6 micrograms/kg) than that of the other samples. On the other hand, no aflatoxin was detected in the control samples that were not inoculated but kept at the same conditions.     

Detection and quantitation of aflatoxin B1 in orange juice by SOS-Chromotest

A new method for the detection and quantitation of aflatoxin B1 in liquids is described. The method is based on the SOS Chromotest, in which damage caused by aflatoxin B1 to the DNA of suitably engineered E. coli induces beta-galactosidase. Aflatoxin B1 developing in orange juice inoculated with spores of Aspergillus parasiticus is detectable equally well by TLC as by the SOS-Chromotest.     

Aflatoxin reduction in corn through field application of competitive fungi.

Soil in corn plots was inoculated with nonaflatoxigenic strains of Aspergillus flavus and A. parasiticus during crop years 1994 to 1997 to determine the effect of application of the nontoxigenic strains on preharvest aflatoxin contamination of corn. Corn plots in a separate part of the field were not inoculated and served as controls. Inoculation resulted in significant increases in the total A. flavus/parasiticus soil population in treated plots, and that population was dominated by the applied strain of A. parasiticus (NRRL 21369). In the years when weather conditions favored aflatoxin contamination (1996 and 1997), corn was predominately colonized by A. flavus as opposed to A. parasiticus. In 1996, colonization by wild-type A. flavus was significantly reduced in treated plots compared with control plots, but total A. flavus/parasiticus colonization was not different between the two groups. A change to a more aggressive strain of A. flavus (NRRL 21882) as part of the biocontrol inoculum in 1997 resulted in a significantly (P < 0.001) higher colonization of corn by the applied strain. Weather conditions did not favor aflatoxin contamination in 1994 and 1995. In 1996, the aflatoxin concentration in corn from treated plots averaged 24.0 ppb, a reduction of 87% compared with the aflatoxin in control plots that averaged 188.4 ppb. In 1997, aflatoxin was reduced by 66% in treated corn (29.8 ppb) compared with control corn (87.5 ppb). Together, the data indicated that although the applied strain of A. parasiticus dominated in the soil, the nonaflatoxigenic strains of A. flavus were more responsible for the observed reductions in aflatoxin contamination. Inclusion of a nonaflatoxigenic strain of A. parasiticus in a biological control formulation for aflatoxin contamination may not be as important for airborne crops, such as corn, as for soilborne crops, such as peanuts.     

Ethylene inhibits aflatoxin biosynthesis in Aspergillus parasiticus grown on peanuts

The filamentous fungi Aspergillus parasiticus and Aspergillus flavus synthesize aflatoxins when they grow on a variety of susceptible food and feed crops. These mycotoxins are among the most carcinogenic naturally occurring compounds known and they pose significant health risks to humans and animals. We previously demonstrated that ethylene and CO2 act alone and together to reduce aflatoxin synthesis by A. parasiticus grown on laboratory media. To demonstrate the potential efficacy of treatment of stored seeds and grains with these gases, we tested ethylene and CO2 for ability to inhibit aflatoxin accumulation on Georgia Green peanuts stored for up to 5 days. We demonstrated an inverse relationship between A. parasiticus spore inoculum size and the level of toxin accumulation. We showed that ethylene inhibits aflatoxin synthesis in a dose-dependent manner on peanuts; CO2 also inhibits aflatoxin synthesis over a narrow dose range. Treatments had no discernable effect on mold growth. These observations support further exploration of this technology to reduce aflatoxin contamination of susceptible crops in the field and during storage.     

Water activity influence on aflatoxin accumulation in corn

The influence of water activity on the production of B1, B2, G1 and G2 aflatoxins in corn has been examined. Viable corn kernels were conditioned at three water activity levels (0.87, 0.90 and 0.97) and inoculated with Aspergillus parasiticus at 30 degrees C. Aflatoxin accumulation was determined at selected times by thin-layer chromatography. For the strain used total aflatoxin accumulation was greater at water activity 0.90 than at 0.87 and 0.97 for the incubation times studied.     

Saccharomyces cerevisiae strains and the reduction of Aspergillus parasiticus growth and aflatoxin B(1) production at different interacting environmental conditions, in vitro

The effect of Saccharomyces cerevisiae RC008 and RC016, previously selected based on their aflatoxin B(1) binding ability and beneficial properties, against Aspergillus parasiticus under different interacting environmental conditions was evaluated. Studies concerning the lag phase, growth rate and aflatoxin B(1) production were carried out in?vitro under different regimes of a (w) (0.95 and 0.99), pH (4 and 6), temperature (25 and 37°C), and oxygen availability (normal and reduced). Both yeast strains showed great antagonistic activity at pH 4, decreasing growth rate compared with the control. The RC008 strain showed the greatest inhibitory activity at all assayed conditions. A. parasiticus produced large amounts of AFB(1) in?vitro. A significant decrease of AFB(1) levels in comparison with the control were observed with yeast interaction. Differences between control and treatment values ranged from 130 to 5400?ng?ml(-1). S. cerevisiae RC008 and RC016 could be considered as effective agents in reducing growth and AFB(1) production at different interacting environmental conditions, related to that found in stored feedstuff. The importance of the present work lies in the search for live strains with both probiotic and biocontrol properties able to prolong the safe storage of feedstuff and exert beneficial properties after animal consumption and which could be included in a novel product for animal feed.     

Novel techniques for controlling the growth of and aflatoxin production by Aspergillus parasiticus in packaged peanuts

The effects of modified atmosphere packaging involving oxygen absorbents, storage temperature and packaging film barrier characteristics on the growth of and aflatoxin production by Aspergillus parasiticus in packaged peanuts was investigated. Mold growth was barely visible in air-packaged peanuts using a high gas barrier film (ASI) while extensive mold growth was observed in peanuts packaged under similar gaseous conditions using a low barrier film (ASIII). Incorporation of an oxygen absorbent (Ageless type S) inhibited mold growth in peanuts packaged in film ASI, while mold growth occurred in peanuts packaged with an absorbent/carbon dioxide generator (Ageless type G) in film ASI and in all absorbent-packaged peanuts in film ASIII. Aflatoxin B₁ production was detected at levels greater than the regulatory limit of 20 ng g^-1 in air-packaged peanuts using film ASI at 20°C and 25°C with the maximum level of aflatoxin (52·95 ng g^-1) being detected in air-packaged peanuts using film ASIII. However, aflatoxin production in all absorbent-packaged peanut samples was less than the regulatory level of 20 ng g^-1 irrespective of the barrier characteristics of the packaging film. Discoloration was more intensive in air-packaged peanuts in film ASIII especially at 25°C and 30°C than those packaged under similar or modified gaseous conditions using film ASI. This study has shown that oxygen absorbent technology is a simple and effective means of controlling the growth of and aflatoxin production by A. parasiticus. However, the effectiveness of these absorbents is dependent on the gas barrier properties of the packaging film surrounding the product.     

Production of aflatoxin B1 and G1 by Aspergillus parasiticus on silica gels.

Silica gels were prepared by acidifying alkaline silicate solutions with phosphoric or tartaric acid. Various combinations of glucose, sucrose, yeast extract, and salts were included in the gels an nutrients. Maximum production of aflatoxins B1 and G1 occurred when silica gel (0.4 to 0.5 cm deep in a petri dish) containing 20% sucrose and 2% yeast extract, and gelled with tartaric acid, was inoculated with approximately 120 to 12000 spores of Aspergillus parasiticus per plate; and plates were incubated at 28 degrees C for 10 days.     

Growth and aflatoxin production by Aspergillus parasiticus in a medium at different pH values and with or without pimaricin

A glucose-yeast extract-salt medium containing 0, 5, 7.5, 10, 15 or 20 micrograms pimaricin/ml with an initial pH of 3.5 or 5.5 was inoculated with Aspergillus parasiticus WB 108 and incubated at 15 degrees or 28 degrees C. The pH, weight of mycelium and amount of aflatoxin produced were determined after 3, 7, and 10 days and after 14, 21, and 30 days when incubation was at 28 degrees or 15 degrees C, respectively. Increasing the concentration of pimaricin in the medium with an initial pH of 5.5 decreased the amounts of aflatoxin B1 and G1 produced after 3 days of incubation. When the initial pH of the medium was 3.5, no growth or toxin production occurred after 3 days of incubation in the medium containing 7.5 micrograms or more of pimaricin/ml. The presence of 20 micrograms of pimaricin/ml inhibited growth and toxin production after 7 days of incubation. When cultures were incubated at 15 degrees C, there was a lag phase which extended from 9 to 16 days, and the amounts of aflatoxin produced decreased with an increasing concentration of pimaricin. Pimaricin did not completely inhibit the growth and aflatoxin production by A. parasiticus. Pimaricin, in combination with a low pH, low temperature or 4% or 6% NaCl, initially caused slow mycelial growth and low toxin production, but the mold overcame the inhibitory effects and produced substantial amounts of mycelium and toxin.     

Assessment of hot peppers for aflatoxin and mold proliferation during storage

Aflatoxin contamination and mold proliferation in three hot pepper hybrids (Sky Red, Maha, and Wonder King) were studied during 5 months of storage at three temperatures (20, 25, and 30°C) and under different packaging conditions (low-density polyethylene bags and jute bags). The presence of aflatoxins in hot pepper samples was determined by high-performance liquid chromatography with a UV-Vis detector. Sampling for analysis of aflatoxins, total mold counts, and Aspergillus counts was carried out at 0, 50, 100, and 150 days of storage. Hot peppers packed in jute bags were more susceptible to aflatoxin contamination than those packed in polyethylene bags; aflatoxin concentrations were 75% higher in peppers stored in jute bags. The effect of storage temperature resulted in aflatoxin concentrations that were 61% higher in hot peppers stored at 25 and 30°C than in those stored at 20°C. Of the three pepper hybrids, Wonder King was more susceptible to aflatoxin contamination, with a maximum of 1.50 μg/kg when packed in jute bags and stored at 25°C for 150 days. However, no sample exceeded the maximum permitted level for total aflatoxins in spices established by European Union regulations (10 μg/kg). Total mold counts and Aspergillus counts increased with storage duration, but all counts were significantly lower in peppers stored in polyethylene bags. A gradual increase in temperature during prolonged storage of hot peppers in combination with aeration may be the main reasons for increases in fungal biomass and Aspergillus proliferation with the subsequent aflatoxin production.     

Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize

Aflatoxin contamination resulting from maize infection by Aspergillus flavus is both an economic and a public health concern. Therefore, strategies for controlling aflatoxin contamination in maize are being investigated. The abilities of eleven naturally occurring atoxigenic isolates in Nigeria to reduce aflatoxin contamination in maize were evaluated in grain competition experiments and in field studies during the 2005 and 2006 growing seasons. Treatments consisted of inoculation of either grains in vials or ears at mid-silking stage in field plots, with the toxigenic isolate (La3228) or atoxigenic isolate alone and co-inoculation of each atoxigenic isolate and La3328. Aflatoxin B(1) + B(2) concentrations were significantly (p < 0.05) lower in the co-inoculation treatments compared with the treatment in which the aflatoxin-producing isolate La3228 was inoculated alone. Relative levels of aflatoxin B(1) + B(2) reduction ranged from 70.1% to 99.9%. Among the atoxigenics, two isolates from Lafia, La3279 and La3303, were most effective at reducing aflatoxin B(1) + B(2) concentrations in both laboratory and field trials. These two isolates have potential value as agents for the biocontrol of aflatoxin contamination in maize. Because these isolates are endemic to West Africa, they are both more likely than introduced isolates to be well adapted to West African environments and to meet regulatory concerns over their use throughout that region.     

Surface binding of aflatoxin B1 by Saccharomyces cerevisiae strains with potential decontaminating abilities in indigenous fermented foods

Saccharomyces cerevisiae constitutes one of the most important microorganisms involved in food fermentations throughout the world. Aflatoxin B(1) binding abilities of S. cerevisiae strains isolated from indigenous fermented foods from Ghana, West Africa were tested in vitro. Results show that aflatoxin binding was strain specific with 7 strains binding 10-20%, 8 strains binding 20-40% and 3 strains binding more than 40% of the added aflatoxin B(1) when grown and incubated under standard conditions. Binding by two of the strains was further characterized. Highest binding capacity was seen with cells collected at the exponential growth phase with the strains A18 and 26.1.11 binding 53.0 and 48.8% of the total toxin respectively and the binding reduced towards the stationary phase. Aflatoxin B(1) binding increased steadily when the cells were incubated with 1 to 20 microg/ml of aflatoxin B(1). Binding was not affected by the cells grown at temperatures ranging from 20 to 37 degrees C, but was significantly reduced at 15 degrees C. Binding seems to be a physical phenomenon with cells treated at 52, 55 and 60 degrees C for 5 and 10 min or 120 degrees C for 20 min binding significantly higher quantities (more than 2-fold in 120 degrees C treated cells) of aflatoxin B(1) than their viable counterpart. Similarly, when the cells were treated with 2 M HCl for 1 h, up to 2-fold increase in binding was observed. The results obtained show that some strains of S. cerevisiae, viable or non-viable, are effective aflatoxin binders and these properties should be considered in the selection of starter cultures for relevant indigenous fermented foods where high aflatoxin level is a potential health risk.     

脂氧合酶与作物黄曲霉毒素污染抗性关系研究进展

曲霉属真菌（Aspergillus）侵染玉米、花生等富含油脂的作物种子后产生的黄曲霉毒素（aflatoxin）具有强致癌作用,严重威胁食品安全和人类健康。脂氧合酶（LOX）及其代谢衍生物在曲霉菌一种子互作中具有重要调节作用。LOX活性的增加可提高植物对细菌、真菌和病毒等病原物的抵抗力,同时由于其催化不饱和脂肪酸代谢生成的脂氧合物如茉莉酸甲酯以及挥发性醛类等物质,可影响黄曲霉菌的生长及黄曲霉毒素的生物合成,因而LOX在作物黄曲霉毒素污染抗性遗传改良中具有潜在的利用价值。本文评述了脂氧合酶及其代谢产物与黄曲霉毒素污染抗性关系的研究进展,旨在从植物一病原菌互作的角度揭示作物黄曲霉毒素污染抗性机制,为下一步的研究提供指导。     

Growth and biosynthesis of aflatoxin by Aspergillus parasiticus in cultures containing nisin

Nisin, 200 or 5000 Reading units/ml, was added to Aspergillus parasiticus cultures. The cultures were incubated at 28 degrees C for 3, 7 or 10 days and analyzed for mycelial dry weight, pH and accumulation of aflatoxin B1 and G1. During the first 3 days of incubation, dry weight, pH decrease and aflatoxin accumulation were suppressed by nisin, when compared with similar values for the nisin-free control. After longer incubation, differences in dry weight nd pH values decreased, whereas accumulation of aflatoxin in the nisin-containing cultures surpassed that of the control.     

Production of aflatoxins on amaranth seeds by Aspergillus spp.

Five varieties of amaranth seeds, cooked and uncooked, were inoculated with toxigenic strains of Aspergillus spp. and the levels of aflatoxins were quantitated. Cooking seeds prior to inoculation increased the level of aflatoxins; aflatoxin levels were the same in all the amaranth varieties inoculated. Aflatoxin level was much lower on amaranth than on rice, corn or winged bean.     

A New Method for Aflatoxin-Free Storage of Agricultural Commodities

Aflatoxin production was observed in the 2 kg lots of peanuts and corn that were stored for 90 days at ambient temperature (28–30°C) and a relative humidity of 100% after infecting with the spores of aflatoxin producing strain Aspergillus parasiticus NRRL 3145. Treatment of the samples with an aqueous solution of 2-chloroethylphosphonic acid prevented aflatoxin formation in both the commodities, whereas, the untreated lots supported aflatoxin formation.     

微生物代谢黄曲霉毒素B1的研究进展

黄曲霉毒素是由黄曲霉、寄生曲霉等真菌产生的次级代谢产物，主要存在于谷类、花生、玉米等农作物中。在我国尤其是南方省份，受高温高湿环境的影响，黄曲霉毒素在农作物上的污染极易发生，造成巨大的经济损失。利用微生物代谢黄曲霉毒素，具有专一、高效、反应条件温和等诸多优点，是目前公认的理想脱毒方法。本文首先简要介绍了黄曲霉毒素B₁的形成过程、结构特征以及毒性基团，接着重点介绍了已报道的能代谢黄曲霉毒素B₁的微生物种类、关键酶以及代谢途径，最后列举了应用基础研究的例子。     

Aflatoxin contents of stored and artificially inoculated cereals and nuts

Aflatoxin contents of cereals and nuts, collected from local markets of NWFP, were determined by thin layer chromatography (TLC). The seeds of these crops were also inoculated with Aspergillus flavus and the aflatoxin content and its relation with the proximate composition of seeds was studied. The effect of storage for different durations of time (2–3 and 12–18 months) on the aflatoxin content of seeds was also assessed. Aflatoxin content of cereals (wheat, maize and rice) ranged from 14 to 45 μg/kg, and that of nuts (almond, walnut and peanut) varied from 5 to 17 μg/kg. The aflatoxin content was within the safe limit (50 μg/kg) recommended by FAO. The aflatoxin content of inoculated seeds was significantly (p < 0.05) increased over control (un-inoculated seeds). This was positively related (r = 0.65) to moisture content of the seeds. However, negative correlation (r = −0.50) existed between aflatoxin and ash contents of the seeds. Protein, fat and total carbohydrate (NFE) contents were not much affected by inoculation. Prolonged storage for 18 months (1.5 years) significantly (p < 0.05) increased aflatoxin contents of seeds compared to short storage periods (2–3 months). It was concluded that aflatoxin content of food should be monitored to ensure food safety. Prolonged storage of cereal and nuts in warm humid condition should be avoided to minimize the risk of aflatoxin contamination.