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 B₁?+?B₂ 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 B₁?+?B₂ reduction ranged from 70.1% to 99.9%. Among the atoxigenics, two isolates from Lafia, La3279 and La3303, were most effective at reducing aflatoxin B₁?+?B₂ 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.     

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 occurrence in milk and supplied concentrates of goat farms of north‐eastern Italy

BACKGROUND: There is little information about the occurrence of aflatoxin M₁ in goat milk. A survey involving 17 dairy goat farms of north‐eastern Italy was completed during 2005 and 2006, in order to evaluate the prevalence of milk contamination and its relationship with type and level of concentrate supplied. RESULTS: 132 concentrate and 85 milk samples were collected during five farm visits and analysed for aflatoxins. Aflatoxin B₁ (AFB₁) was > 0.1 µg kg^?1 in two‐thirds of the feeds and > 5 µg kg^?1 in nine. Contamination was higher in maize than in other pure feeds (median: 0.8 versus 0.1 µg kg^?1); complementary feeds showed intermediate values. Aflatoxin M₁ (AFM₁) was > 3 ng kg^?1 in one‐third of milks and > 25 ng kg^?1 in three. All the milk samples were below EU statutory limits. The farm ranks for milk AFM₁ level and the peak of concentrate AFB₁ contamination were significantly correlated (0.642). CONCLUSIONS: Risk to human health was generally found to be absent, with only a few cases involving feed contamination to be monitored. The main aflatoxin risk for goat milk could arise from maize and maize‐based concentrates in the more intensive breeding conditions. Copyright © 2008 Society of Chemical Industry     

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.

     

Aflatoxin contamination in mustard (Brassica juncea) in relation to agronomic practices

Aflatoxin contamination in two varieties of mustard (Brassica juncea L), Varuna and BR-40, sown on three planting dates (1, 15 and 30 November) under two different cropping patterns, viz monocropping and mixed cropping (along with the UP-262 variety of wheat), was examined in rabi (winter) crops of 1987–1988 and 1988–1989. Analysis of variance showed a significant effect of planting date and cropping pattern as well as a combined effect of planting date × cropping pattern. Delayed planting (30 November) resulted in a very high level of aflatoxin contamination. The level of aflatoxin was low when mustard was cultivated along with wheat (mixed cropping). Varuna was more susceptible to aflatoxin than BR-40. The maximum number of fungi was recorded in mixed cropping samples.     

Correlation and classification of single kernel fluorescence hyperspectral data with aflatoxin concentration in corn kernels inoculated with Aspergillus flavus spores

The objective of this study was to examine the relationship between fluorescence emissions of corn kernels inoculated with Aspergillus flavus and aflatoxin contamination levels within the kernels. Aflatoxin contamination in corn has been a long-standing problem plaguing the grain industry with potentially devastating consequences to corn growers. In this study, aflatoxin-contaminated corn kernels were produced through artificial inoculation of corn ears in the field with toxigenic A. flavus spores. The kernel fluorescence emission data were taken with a fluorescence hyperspectral imaging system when corn kernels were excited with ultraviolet light. Raw fluorescence image data were preprocessed and regions of interest in each image were created for all kernels. The regions of interest were used to extract spectral signatures and statistical information. The aflatoxin contamination level of single corn kernels was then chemically measured using affinity column chromatography. A fluorescence peak shift phenomenon was noted among different groups of kernels with different aflatoxin contamination levels. The fluorescence peak shift was found to move more toward the longer wavelength in the blue region for the highly contaminated kernels and toward the shorter wavelengths for the clean kernels. Highly contaminated kernels were also found to have a lower fluorescence peak magnitude compared with the less contaminated kernels. It was also noted that a general negative correlation exists between measured aflatoxin and the fluorescence image bands in the blue and green regions. The correlation coefficients of determination, r ², was 0.72 for the multiple linear regression model. The multivariate analysis of variance found that the fluorescence means of four aflatoxin groups, <1, 1–20, 20–100, and ≥100 ng g^?1 (parts per billion), were significantly different from each other at the 0.01 level of alpha. Classification accuracy under a two-class schema ranged from 0.84 to 0.91 when a threshold of either 20 or 100 ng g^?1 was used. Overall, the results indicate that fluorescence hyperspectral imaging may be applicable in estimating aflatoxin content in individual corn kernels.     

Validation of a lateral flow test (MRLAFMQ) for the detection of aflatoxin M1 at 50 ng l−1 in raw commingled milk

Aflatoxin M₁ contamination in dairy products is a risk when feedstuff contaminated with aflatoxin B₁ produced by moulds is consumed by milk-producing animals. Milk can be screened for aflatoxin M₁ at the European Union maximum limit of 50 ng l^?1 by a lateral flow test, the MRLAFMQ (Aflatoxin M1) Test. The method takes 15 min with no milk dilution or a sample preparation step. The lateral flow assay was validated at the Technology and Food Science Unit of the Institute for Agricultural and Fisheries Research (ILVO-T&V) according to European Union guidelines using fortified raw milk samples. A detection capability of 50 ng l^?1 was demonstrated with a false negative rate lower than 2% at 50 ng l^?1 and a false positive rate of less than 0.3%. Quantitative readings had a mean bias of +2 to 6 ng l^?1 at 50 ng l^?1 with a standard deviation of 5–8 ng l^?1. Based on the validation results, the test could be considered appropriate for milk screening prior to milk unload at dairies.     

Aflatoxins in sunflower and safflower seeds from Iran

Aflatoxin content of 173 sunflower and safflower seeds was determined by HPLC with immunoaffinity column (IAC) clean-up and fluorometric detection. Aflatoxin B₁ contamination was found in 111 samples: in 8 of the sunflower seed samples (16%) at a mean level of 40.68?ng?g^?1 and in 103 safflower seed samples (83.7%) at a mean level of 2.81?±?0.44?ng?g^?1. In 5 sunflower seed samples and 1 safflower seed sample, aflatoxin B₁ levels were higher than the maximum levels of AFB₁ under Iran regulations (5?ng?g^?1). Aflatoxin B₁ levels in 5 sunflower and 2 safflower seed samples were higher than the European Union maximum limit (2?ng?g^?1).     

乳及乳制品中黄曲霉毒素M1污染及检测技术研究进展

乳及乳制品中的黄曲霉毒素M_1是黄曲霉毒素B1的羟基化衍生物,对肝脏有致畸和致癌作用。随着人们生活水平的提高,乳及乳制品中黄曲霉毒素M_1污染也引起越来越多的关注。本文从不同品种、季节及地域分析乳及乳制品中黄曲霉毒素M_1污染水平,并综述了液相色谱法和免疫法的检测技术研究进展。     

Mycoflora and aflatoxin contamination in shelled pistachio nuts

A total of 143 pistachio nut samples collected during harvest, storage and processing were examined for mould growth and aflatoxin production. The mould count was in the range of 10³?10⁴ cfu g^?1 and 10⁵?10⁶ cfu g^?1 for the harvest and storage samples, respectively. The growth of Aspergillus flavus was 38-5-39-5% on the surface of the shells and 6–16% on the kernels without aflatoxin production. The contamination level of A flavus varied among samples collected from different regions. Peeling off the soft shell of pistachio nuts by hand reduced the contamination risk of A flavus to kernels. The predominant flora on stored pistachio nuts were Aspergillus, Penicillium, Cladosporium, Rhizopus, while the genera of Ulocladium, Trichothecium, Aureobasidium and Eurotium were less frequent. Thirty-five percent of the A flavus isolates produced aflatoxins on synthetic media.     

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.     

An effective self-control strategy for the reduction of aflatoxin M1 content in milk and to decrease the exposure of consumers

The study reports the results of testing the sensitivity of an early warning sampling plan for detecting milk batches with high aflatoxin AFM₁ concentration. The effectiveness of the method was investigated by the analysis of 9017 milk samples collected in Italian milk processing plants that applied control plans with different action limits (AL). For those milk processing plants where 30 ng kg^?1 AL has been applied, the AFM₁ contamination was significantly lower at or above the 95th percentile of the milk samples when compared with plants that used 40 ng kg^?1 AL. The results show that the control plan can be used effectively for early warning of occurrence of high AFM₁ contamination of milk and to carry out pro-active measures to limit the level of contamination. Estimation of dietary exposure was also carried out, based on the aflatoxin M₁ content of the milk samples and on Italian food consumption data. Estimated Daily Intakes (EDI) and Hazard Indices (HI) were calculated for different age groups of the population. HIs show that no adverse effects are expected for the adult population, but in the case of children under age three, the approximate HI values were considerably higher. This underlines the importance of the careful monitoring and control of aflatoxin M₁ in milk and dairy products.     

Selected plant essential oils and their main active components,a promising approach to inhibit aflatoxigenic fungi and aflatoxin production in food

Recent research has showed that Aspergillus flavus and Aspergillus parasiticus are aflatoxigenic species that can become very competitive in the framework of climate change. Aflatoxins show carcinogenic, mutagenic, immunotoxic and teratogenic effects on human and animals. Effective and sustainable measures to inhibit these species and aflatoxins in food are required. Origanum vulgare and Cinnamomum zeylanicum essential oils (EOs) and their major active constituents, carvacrol and cinnamaldehyde, respectively, were assayed for inhibiting these species and aflatoxin production in maize extract medium under different environmental conditions. Doses of 10–1000 mg l^?1 were assayed and the effective doses for 50 (ED₅₀) and 90% (ED₉₀) growth inhibition were determined. The ED₅₀ of cinnamaldehyde, carvacrol, oregano EO, and cinnamon EO against A. flavus were in the ranges 49–52.6, 98–145, 152–505, 295–560 mg l^?1 and against A. parasiticus in the ranges 46–55.5, 101–175, 260–425 and 490–675 mg l^?1, respectively, depending on environmental conditions. In A. flavus treatments ED₉₀ were in the ranges 89.7–90.5, 770–860 and 820–>1000 mg l^?1 for cinnamaldehyde, carvacrol and cinnamon EO, and in A. parasiticus treatments in the ranges 89–91, 855–>1000 and 900–>1000 mg l^?1, respectively. ED₉₀ values for oregano EO against both species were >1000 mg l^?1. Growth rates of both species were higher at 37 than at 25°C and at 0.99 than at 0.96 a_w. Aflatoxin production was higher at 25 than at 37°C. Stimulation of aflatoxin production was observed at low doses except for cinnamaldehyde treatments. The effectiveness of EOs and their main constituents to inhibit fungal growth and aflatoxin production in contact assays was lower than in vapour phase assays using bioactive EVOH-EO films previously reported.     

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.     

Aflatoxin and Cyclopiazonic Acid Production by Aspergillus flavus Isolated from Contaminated Maize

Seven truck-loads of maize were tested for mycotoxin contamination. Aflatoxin was identified in all 7 at concentrations from 3 ng/g-501 ng/g (aflatoxin B₁+ B₂). Cyclopiazonic acid was identified in 4 loads with concentrations from 25-250 ng/g. Deoxynivalenol was found in 4 of 5 loads tested, over a range of 46-676 ng/g. Ninteeen isolates of Aspergillus flavus from the samples were tested for ability to accumulate cyclopiazonic acid and aflatoxin in liquid culture. Fourteen produced cyclopiazonic acid (0.5-135 μg/mL), 12 produced aflatoxin (0.01-0.70 μg/mL, aflatoxin B₁+ B₂), and one aflatoxin-producing isolate did not produce cyclopiazonic acid.     

Delivery systems for biological control agents to manage aflatoxin contamination of pre-harvest maize

While soil application of a competitive non-toxigenic Aspergillus flavus strains is successful in reducing aflatoxin contamination in certain crops, direct application to aerial reproductive structures could be more effective for maize. A sprayable, clay-based water-dispersible granule formulation was developed to deliver non-toxigenic A. flavus strain K49 directly to maize ears. The efficacy of the K49 water-dispersible granule in mitigating aflatoxin in maize (Zea mays L.) was evaluated. Field studies were conducted to compare K49 colonization and effectiveness in reducing aflatoxin contamination when applied either as a soil inoculant or as a directed spray in plots infested with toxigenic strain F3W4. Fifty percent of non-toxigenic A. flavus was recovered from non-treated controls and from plots soil inoculated with K49 on wheat. In spray treatments with formulated or unformulated K49 conidia, over 90% of A. flavus recovered was non-toxigenic. Soil-applied K49 reduced aflatoxin contamination by 65% and spray applications reduced contamination by 97%. These findings suggest direct spray application of non-toxigenic A. flavus strains may be better than soil inoculation at controlling maize aflatoxin contamination and that a water-dispersible granule is a viable delivery system for maintaining viability and efficacy of the biological control agent, K49.     

Volatile trans-2-hexenal, a soybean aldehyde, inhibits Aspergillus flavus growth and aflatoxin production in corn

Abstract: Trans‐2‐hexenal, a volatile aldehyde, is produced by soybean (Glycine max [L.] Merr) and other plants via the lipoxygenase pathway. In vitro tests showed it significantly (P < 0.001) reduced Aspergillus flavus germinating conidial viability at 10 μM, with approximately 95% viability reduction observed at 20 μM. The viability of nongerminated conidia was not reduced. To test the effectiveness of this volatile to prevent fungal growth in stored corn, trans‐2‐hexenal was pumped intermittently into glass jars containing corn. Experiments were performed to determine the ability of 2 different pump cycle time‐courses to prevent A. flavus growth on sterile corn (23% moisture). Intermittently (30‐min pumping period) over 7 d, this volatile was pumped through 350 g of corn kernels inoculated with 1 mL of 3 × 10⁴ conidia of A. flavus. Controls consisted of (1) sterile corn, (2) corn inoculated with A. flavus with no pumped air, and (3) corn inoculated with A. flavus with intermittently pumped air. Aflatoxin B₁ (AFB₁), viability counts, and aldehyde concentration in the headspace were performed in each experiment. To determine whether an increased time period between volatile pumping would prevent A. flavus growth, a 2nd series of experiments were performed that were similar to the 1st series except that trans‐2‐hexenal (only) was pumped for a 30‐min period every 12 h. Experiments were performed 3 times for each time course. Both experiments showed that intermittent pumping of volatile trans‐2‐hexenal significantly (P < 0.001) prevented A. flavus growth and aflatoxin B₁ production over a 7‐d period. Practical Application: Results from this study indicate that intermittent pumping of volatile trans‐2‐hexenal could be used to protect stored corn from A. flavus growth and aflatoxin contamination.     

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     

Colonisation and aflatoxin formation by Aspergillus spp. on brown rices differing in endosperm properties

Aspergillus flavus Link and A. parasiticus Speare differed in sporulation and aflatoxin B₁ formation on 15 brown rices differing in starch properties and protein content. There was no significant correlation between the degree of spore formation by the fungi and the amount of aflatoxin they produced in the rice samples. Differences in sporulation and aflatoxin production were not correlated with the dimensions (weight and hardness) of brown rice nor with the protein content, alkali spreading value, amylose content and gel viscosity of milled rice. Aflatoxin B₁ was mainly concentrated in the bran layers. Histological examination revealed that the hyphae of A. flavus entered through cracks in the periderm and penetrated the endosperm.     

Aflatoxin production in three selected samples of triticale,wheat and rye grown in Argentina

Little is known about mycotoxin contamination of triticale, a hybrid resulting from crossing wheat and rye. The purpose of the present work was to evaluate triticale as a substrate for aflatoxin accumulation in comparison with its parents. Aflatoxin (B₁, B₂, G₁ and G₂) accumulation curves were obtained for the three substrates inoculated with Aspergillus parasiticus NRRL 2999 and incubated at 25 °C and water activity 0.925 for 10 weeks. Wheat and triticale were poor substrates for aflatoxin production. Rye was more prone than the other substrates to fast colonisation by A parasiticus and accumulated larger aflatoxin quantities over the whole incubation period. The maximum aflatoxin concentration in rye (11 840 µg kg⁻¹) was significantly larger (p < 0.05) than those obtained in wheat (2150 µg kg⁻¹) and triticale (2850 µg kg⁻¹). © 2000 Society of Chemical Industry