Food additives and contaminants最新文献

Fungi belonging to Aspergillus section Flavi are of great economic importance in the United States due to their ability to produce toxic and carcinogenic aflatoxins in agricultural commodities. Development of control strategies against A. flavus and A. parasiticus, the major aflatoxin-producing species, is dependent upon a basic understanding of their diversity in agricultural ecosystems. This review summarizes our current knowledge of species and population diversity in the United States in relation to morphology, mycotoxin production and genetic characters. The high genetic diversity in populations of aflatoxigenic fungi is a reflection of their versatile habits in nature, which include saprotrophic colonization of plant debris in soil and parasitism of seeds and grain. Genetic variation within populations may originate from a cryptic sexual state. The advent of intensive monoculture agriculture not only increases population size but also may introduce positive selective pressure for aflatoxin production due to its link with pathogenicity in crops. Important goals in population research are to determine how section Flavi diversity in agricultural ecosystems is changing and to measure the direction of this evolution.

Aflatoxins are toxic and carcinogenic polyketide metabolites produced by fungal species, including Aspergillus flavus and A. parasiticus. The biosynthesis of aflatoxins is modulated by many environmental factors, including the availability of a carbon source. The gene expression profile of A. parasiticus was evaluated during a shift from a medium with low concentration of simple sugars, yeast extract (YE), to a similar medium with sucrose, yeast extract sucrose (YES). Gene expression and aflatoxins (B1, B2, G1, and G2) were quantified from fungal mycelia harvested pre- and post-shifting. When compared with YE media, YES caused temporary reduction of the aflatoxin levels detected at 3-h post-shifting and they remained low well past 12 h post-shift. Aflatoxin levels did not exceed the levels in YE until 24 h post-shift, at which time point a tenfold increase was observed over YE. Microarray analysis comparing the RNA samples from the 48-h YE culture to the YES samples identified a total of 2120 genes that were expressed across all experiments, including most of the aflatoxin biosynthesis genes. One-way analysis of variance (ANOVA) identified 56 genes that were expressed with significant variation across all time points. Three genes responsible for converting norsolorinic acid to averantin were identified among these significantly expressed genes. The potential involvement of these genes in the regulation of aflatoxin biosynthesis is discussed.

Aflatoxins are toxic and carcinogenic polyketide metabolites produced by certain fungal species, including Aspergillus flavus and A. parasiticus. Many internal and external factors, such as nutrition and environment affect aflatoxin biosynthesis; therefore, we analyzed the transcriptome of A. flavus using expressed sequence tags (ESTs) from a normalized cDNA expression library constructed from mycelia harvested under several conditions. A total of 7218 unique ESTs were identified from 26,110 sequenced cDNA clones. Functional classifications were assigned to these ESTs and genes, potentially involved in the aflatoxin contamination process, were identified. Based on this EST sequence information, a genomic DNA amplicon microarray was constructed at The Institute for Genomic Research (TIGR). To identify potential regulatory networks controlling aflatoxin contamination in food and feeds, gene expression profiles in aflatoxin-supportive media versus non-aflatoxin-supportive media were evaluated in A. flavus and A. parasiticus. Genes consistently expressed in several aflatoxin-supportive media are reported.

Aflatoxins (AFs) are carcinogenic secondary metabolites of Aspergillus parasiticus. In previous studies, non-toxigenic A. parasiticus sec- (for secondary metabolism negative) variants were generated through serial transfer of mycelia from their toxigenic sec+ (for secondary metabolism positive) parents for genetic and physiological analysis for understanding regulation of AF biosynthesis. Previous studies have shown no difference in the DNA sequence of aflR, a positive regulator of AF production, in the sec+ and sec- strains. In this study, AflJ, another positive regulator of AF production, laeA, a global regulator of secondary metabolism, and the intergenic region between aflR and aflJ, were analysed to determine if they play a role in establishment of the sec- phenotype. The study showed that while this sequence identity extended to the aflJ as well as the aflJ-aflR intergenic region, expression of aflR in the sec- strain was several fold lower than that observed in the sec+ strain, while aflJ expression was barely detectable in the sec- strain. Western blot analysis indicated that despite AflR protein being present in the sec- strain, no toxin production resulted. Introduction of a second copy of aflR into the sec- strain increased aflR expression, but did not restore AF production. Lastly, reverse transcription-PCR analysis revealed that laeA was expressed in both sec+ and sec- strains. These results suggest that although aflR, aflJ and laeA are necessary for AF production, they are not sufficient. We propose that the aflR and aflJ expression may be regulated by element(s) downstream from laeA or from pathways not influenced by laeA.

Polyketides are a structurally diverse class of secondary metabolites produced by bacteria, fungi, plants and animals. The fungal genus Fusarium includes agronomically important plant pathogenic and mycotoxin-producing species and produces numerous polyketides. The study further characterized a polyketide synthase-encoding gene (PKS3 = PGL1) that was previously identified in F. graminearum and F. verticillioides. Disruption of the F. verticillioides PGL1 indicated that it is required for the production of the dark pigment in perithecial walls, as previously shown in F. graminearum. A third PGL1 orthologue was identified in the genomic sequence of N. haematococca (anamorph F. solani f. sp. pisi). Analysis of the carboxy-terminal end of the deduced PGL1 protein indicated that it had a functional domain related to dehydrogenases/reductases that is sometimes present in non-ribosomal peptide synthetases. Comparison of the genomic regions flanking PGL1 in F. graminearum, F. verticillioides and N. haematococca revealed that the extent of gene synteny in this region was greater between F. graminearum and F. verticillioides than between either of these species and N. haematococca. Southern blot analysis indicated that PGL1 occurs widely within the genus Fusarium including species with no known sexual stage.

Fusarium proliferatum is a major cause of maize ear rot and fumonisin contamination and also can cause wheat kernel black point disease. The primary objective of this study was to determine whether nine F. proliferatum strains from wheat from Nepal can cause black point and fumonisin contamination in wheat kernels. For comparison, the study included three Fusarium strains from US maize. In test 1, all the strains but one produced significant symptoms of kernel black point; two strains decreased kernel yield; and four strains contaminated kernels with fumonisins B(1), B(2) and B(3) as determined by liquid chromatography-mass spectroscopy. Strain Ggm202 from Nepal, which produced the highest levels of fumonisins (mean = 49 microg g(-1)) on five wheat cultivars in test 1, was confirmed to produce fumonisins (mean = 38 microg g(-1)) on two cultivars in test 2. The data indicate a potential for fumonisin contamination of wheat infected with F. proliferatum.

Single-stranded conformational polymorphism (SSCP) analysis for genetic diversity studies has been widely applied to detect indirectly sequence differences up to a single base in amplified DNA fragments of the same length, representing an alternative to gene sequencing. In this study SSCP analysis was used to detect sequence variations contained in an about 180-bp region of the calmodulin gene in order to identify Aspergillus section Nigri species. The method described shows that fluorescence-based SSCP analysis by capillary electrophoresis is cheaper and faster than direct sequencing, and suitable for computer-assisted analyses allowing discrimination between the Aspergillus species belonging to the Nigri section: A. aculeatus, Aspergillus 'atypic uniseriate', A. brasiliensis, A. carbonarius, A. ellipticus, A. foetidus, A. heteromorphus, A. ibericus, A. japonicus, A. niger, and A. tubingensis.

This review focuses on recent developments in immunochemical methods for detection of mycotoxins, with a particular emphasis on simultaneous multiple analyte determination. This includes high-throughput instrumental analysis for the laboratory environment (microtitre plate enzyme-linked immunoabsorbant assay (ELISA), different kinds of immunosensors, fluorescence polarization immunoassay, and capillary electrophoretic immunoassay), as well as rapid visual tests for on-site testing (lateral-flow, dipstick, flow-through and column tests). For each type of immunoassay, perspectives for multiple analyte application are discussed and examples cited.

A multi-analyte method for the liquid chromatography-tandem mass spectrometric determination of mycotoxins in crude grain extracts without clean-up has been applied to the analysis of spelt, rice and barley. Method performance characteristics were determined after spiking blank samples at multiple levels and were found to be comparable for all investigated matrices as regards linearity (linear calibration functions were obtained for all analyte/matrix combinations except for moniliformin), precision (coefficient of variations <6%) and sensitivity. Matrix-induced signal suppression/enhancement was studied in detail and varied significantly between the investigated matrices, as well as between individual samples (relative standard deviation was as high as 40% within three rice varieties) and individual toxins. It was concluded that a reliable quantitative analysis using matrix-matched calibration requires careful consideration of the model matrix, which should match the investigated samples as close as possible.

It is demonstrated that, in fungal cells grown in synthetic media, the Apyap1 gene is implicated in the modulation of aflatoxin biosynthesis following the perturbation of redox balance. This study suggests that an association between oxidative stress and aflatoxin biosynthesis also occurs in maize seeds. We used DeltaApyap1, a strain in which the gene Apyap1 was disrupted, to verify whether this oxidative stress-related transcription factor, by affecting cell redox balance, can have a role in the modulation of aflatoxin synthesis. The amount of hydroperoxides (ROOH) produced by wild type (WT) and DeltaApyap1, both grown in potato dextrose broth, was assayed in the filtrate. In maize seeds (30 g), inoculated with WT and DeltaApyap1conidia and incubated at 30 degrees C for 15 days, lipoxygenase activity (LOX), lipoperoxides (LOOH) production, fungal growth and aflatoxin biosynthesis was analysed. It was observed that DeltaApyap1 released more hydroperoxides in the culture media and more aflatoxins in seeds, possibly through stronger stimulation of LOX, which, in turn led to greater LOOH production in the seeds. On the basis of the results, a hypothesis regarding strategies to control aflatoxin synthesis is formulated.