Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues

The luncheon meat samples analyzed, which were produced locally by the two main luncheon meat producing companies in Egypt were relatively highly contaminated either by moulds and yeasts in general, aflatoxigenic species and aflatoxin residues in particular. The most frequently encountered fungi from the samples were yeasts, Aspergillus niger, A. flavus, Penicillium chrysogenum, Rhizopus stolonifer, Mucor circinelloides. Less common were Cladosporium sphaerospermum, Alternaria alternata, Mycosphaerella tassiana, P. aurantiogriseum and P. oxalicum. The most important aflatoxigenic species, A. flavus, was isolated frequently. It was 10% of the total fungal isolates from both samples of the two companies. Seven luncheon meat samples out of 50 analyzed were positive for aflatoxin B₁ or B₁ and G₁, while all samples were negative for aflatoxins B₂, G₂, M₁ and M₂. Aflatoxin B₁ was detected only in 4 and 3 samples out of 25 analyzed from each of company A and B, respectively. The highest detectable level, 11.1 ppb, was recorded in a sample from company B and the least, 0.5 ppb, in a sample from company A. Aflatoxin G₁, at concentration of 3.2 ppb, was detected in only one sample of the aflatoxin B₁ – contaminated 3 samples of company B: this sample also had the highest level of aflatoxin B₁. Some luncheon meat samples had higher numbers of aflatoxigenic A. flavus than others, however these samples were negative for aflatoxins. The hazardous potential of such contamination will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Emericella astellata, a new producer of aflatoxin B, B and sterigmatocystin

AIMS: To report on aflatoxin B(1) and B(2) production from a species of Emericella. METHODS AND RESULTS: Aflatoxins and sterigmatocystin were determined by high-pressure liquid chromatography (HPLC) with diode array detection and confirmed by HPLC with mass spectrometry detection. Among 30 known species of Emericella only one species produced aflatoxin. Strains originating from the same geographical source material had different patterns of aflatoxin and sterigmatocystin production on different media, indicating that epigenetic factors may be involved in the regulation of aflatoxin production. However, two cultures from the same original genet were very similar. CONCLUSIONS: Emericella astellata can produce small amounts of sterigmatocystin and aflatoxin B(1) and B(2). SIGNIFICANCE AND IMPACT OF THE STUDY: Emericella has been used extensively in genetic studies and therefore the isolates producing aflatoxin can be used to elucidate the genetic, evolutionary and maybe ecological role of aflatoxins using molecular genetic methods.     

Characterization of an echinocandin B-producing strain blocked for sterigmatocystin biosynthesis reveals a translocation in the stcW gene of the aflatoxin biosynthetic pathway

Echinocandin B (ECB), a lipopolypeptide used as a starting material for chemical manufacture of the anti-Candida agent LY303366, is produced by fermentation using a strain of Aspergillus nidulans. In addition to ECB, the wild-type strain also produces a significant level of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Characterization of a mutant designated A42355-OC-1 (OC-1), which is blocked in ST biosynthesis, was the result of a chromosomal translocation. The chromosomal regions containing the breakpoints of the translocation were isolated and DNA sequencing and PCR analysis of the chromosomal breakpoints demonstrated the translocation occurred within the stcW gene of the ST biosynthetic pathway, resulting in disruption of the open reading frame for this gene. Biochemical feeding studies indicate the involvement of this gene product in the conversion of averufin to 1-hydroxy versicolorone. This work demonstrates an effective synergy between classical strain improvement methods and molecular genetics. Journal of Industrial Microbiology & Biotechnology (2000) 25, 333–341. Received 27 April 2000/ Accepted in revised form 25 November 2000     

Effect of Inoculum Rate of Biological Control Agents on Preharvest Aflatoxin Contamination of Peanuts

Studies were conducted during 1994 and 1995 in the environmental control plot facility at the National Peanut Research Laboratory to determine the effect of different inoculum rates of biological control agents on preharvest aflatoxin contamination of Florunner peanuts. Biocontrol agents were nontoxigenic color mutants ofAspergillus flavusandAspergillus parasiticusthat were grown on rice for use as soil inoculum. Three replicate plots (4.0 × 5.5 m) were treated with 0, 2, 10, and 50 g/m of row (0, 20, 100, and 500 lb/acre, respectively) of an equal mixture of the color mutant-infested rice in 1994, and the same plots were retreated in 1995. Aflatoxin concentrations were determined by high performance liquid chromatographic analysis of all peanuts. Treatment means for total kernels in 1994 were 337.6, 73.7, 34.8, and 33.3 ppb for the 0, 2, 10, and 50 g/m treatments, respectively. Regression analysis indicated a trend toward lower aflatoxin concentrations with increasing rates of inoculum (R²= 0.40;P< 0.05). For the same repeated treatments in 1995 aflatoxin concentrations in total kernels averaged 718.3, 184.4, 35.9, and 0.4 ppb. Regression analysis revealed a stronger relationship between inoculum rate and aflatoxin concentrations (R²= 0.66;P< 0.05) in the second year of treatment. Compared with untreated controls, the 2, 10, and 50 g/m treatments produced respective reductions in aflatoxin of 74.3, 95.0, and 99.9% in the second year. The data indicated not only a treatment-related effect, but also that a higher degree of control might be achieved when plots or fields are retreated with biocontrol agents in subsequent years.     

β-Carotene inhibition of aflatoxin biosynthesis amongAspergillus flavus genotypes from Illinois corn

Thirty-nineAspergillus flavus genotypes (DNA fingerprinting) isolated from corn grown in a field near Kilbourne, Illinois were evaluated for their sensitivity to β-carotene (50 μg/ml) inhibition of aflatoxin B₁ biosynthesis. Inhibition of aflatoxin was greater than 90% for 28 of the genotypes and >70% for 38 of the 39 genotypes. FiveA. flavus strains (4 fingerprint groups) isolated from molded raw peanuts, NRRL 3239, NRRL 3357, NRRL 6514, NRRL 6515 and NRRL 13135, produced greater quantities of aflatoxin than all 39 genotypes isolated from corn, and were less sensitive to β-carotene inhibition.Aspergillus flavus NRRL 3357 is commonly used as inoculum in variety trials for aflatoxin resistance. Isolate identity and sensitivity to potential inhibitors in corn can be critical in assessing corn resistance to aflatoxin.     

Sex-determined differential mortality of milkweed bugs, Oncopeltus fasciatus (hemiptera), to aflatoxins

Studies on the aflatoxins, toxic metabolites of Aspergillus flavus and A. parasiticus, have involved test systems ranging from cell cultures to laboratory animals. This work reports on the differential response by sex of Oncopeltus fasciatus to aflatoxin B₁ (AFB₁). Young adult milkweed bugs were chosen randomly from our stock colony and housed in glass culture jars. Triplicate sets of experimental animals were fed 5 μg/ml of AFB₁ in their liquid diet. The first death for the experimental females occurred at day 4, and at 10 days for the experimental males. A 50% lethality level for experimental females developed by day 8. Males subjected to the same concentration achieved a 50% lethality level at day 24. For the females the LD₅₀ occurred after consuming 0.49 μg/ml of AFB₁. The results indicate that adult female milkweed bugs were hypersensitive to AFB₁ as compared to adult males. This organism is more sensitive than the American cockroach and less sensitive than the fruitfly, housefly, and honeybee to toxic aflatoxicosis. Even the female is not sufficiently sensitive to rate highly as a bioassay organism for AFB₁. The extreme difference in mortality between the sexes is significant, unusual, and unexplained.     

Analysis of single nucleotide polymorphisms in three genes shows evidence for genetic isolation of certain Aspergillus flavus vegetative compatibility groups

Genetic exchange by asexual filamentous fungi is presumed to be limited to isolates in the same vegetative compatibility group (VCG). To evaluate genetic isolation of Aspergillus flavus due to vegetative incompatibility, three gene regions were chosen that contained closely spaced nucleotides that were polymorphic among some of the six VCGs examined. A member of each VCG was collected from five regions across the southern United States. Isolates belonging to the same VCG had similar sets of single nucleotide polymorphisms regardless of isolate origin. The six VCGs formed four genetically distinct groups. Although recombination between certain pairs of VCGs could not be excluded, none was found for YV36, the VCG that includes the atoxigenic A. flavus isolate currently used to mitigate aflatoxin contamination in cotton in Arizona.     

Method for monitoring deletions in the aflatoxin biosynthesis gene cluster of Aspergillus flavus with multiplex PCR

The report presents a rapid, inexpensive and simple method for monitoring indels with influence on aflatoxin biosynthesis within Aspergillus flavus populations. PCR primers were developed for 32 markers spaced approximately every 5 kb from 20 kb proximal to the aflatoxin biosynthesis gene cluster to the telomere repeat. This region includes gene clusters required for biosynthesis of aflatoxins and cyclopiazonic acid; the resulting data were named cluster amplification patterns (CAPs). CAP markers are amplified in four multiplex PCRs, greatly reducing the cost and time to monitor indels within this region across populations. The method also provides a practical tool for characterizing intraspecific variability in A. flavus not captured with other methods.

Significance and Impact of the Study

Aflatoxins, potent naturally‐occurring carcinogens, cause significant agricultural problems. The most effective method for preventing contamination of crops with aflatoxins is through use of atoxigenic strains of Aspergillus flavus to alter the population structure of this species and reduce incidences of aflatoxin producers. Cluster amplification pattern (CAP) is a rapid multiplex PCR method for identifying and monitoring indels associated with atoxigenicity in A. flavus. Compared to previous techniques, the reported method allows for increased resolution, reduced cost, and greater speed in monitoring the stability of atoxigenic strains, incidences of indel mediated atoxigenicity and the structure of A. flavus populations.     

Contamination of Groundnut in South‐Western Nigeria by Aflatoxigenic Fungi and Aflatoxins in Relation to Processing

Groundnut is commonly consumed in its roasted form by many Nigerians. This study was therefore conducted to determine the levels of aflatoxin in roasted groundnut retailed in south‐western Nigeria with a view to assessing the fitness of the processed nut for human consumption. The effects of roasting and de‐coating as alternative methods for reducing the ‘aflatoxin scare’ in the nut were further assessed on aflatoxigenic fungal load and aflatoxin content of the nuts. Forty‐eight samples of retailed raw and roasted groundnut were collected and assessed by mycological and thin‐layer chromatographic analysis for changes in aflatoxigenic fungal population and aflatoxin concentration, respectively. Consequently, 480 isolates of the Aspergillus section Flavi group, A. flavus L strain (n = 410), A. tamarii (n = 56), A. parasiticus (n = 7) and A. parvisclerotigenus (n = 7), were recovered from all samples. Aflatoxigenic isolates of A. flavus L strain (58.8%) had a significantly (P < 0.05) higher incidence than the non‐aflatoxigenic isolates (41.2%). Aflatoxins were detected in 43 (89.6%) of the samples. Approximately 25% of all samples exceeded the 20 ng/g limit for aflatoxin B₁ (AFB₁) adopted by the National Agency for Food and Drug Administration and Control while 83 and 79% of all samples contained AFB₁ and total aflatoxins above the European Union limits of 2 and 4 ng/g, respectively. Aflatoxin concentrations in the raw and coated samples were as much as five times higher than those in the roasted and de‐coated nuts, respectively. However, no significant difference was recorded between aflatoxin levels in the coated and de‐coated samples. This study has shown that roasting of groundnut and testa removal (de‐coating) are effective processing interventions that can significantly lower aflatoxin quantities in the kernels, thus making it fit for human consumption.     

Divergent regulation of aflatoxin production at acidic pH by two <Emphasis Type="Italic">Aspergillus</Emphasis> strains

Production of aflatoxins (AF) by Aspergillus flavus and A. parasiticus is known to occur only at acidic pH. Although typical A. flavus isolates produced more AF as the external pH became increasingly acidic, an atypical strain from West Africa produced less. The lower AF production was not well correlated with decreases in expression of the aflatoxin pathway regulatory gene, aflR, or of two other biosynthesis genes.