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Background: Meat-products are considered an enriched media for mycotoxins. This study aimed to investigate the prevalence of toxigenic Aspergillus species in processed meat samples, HPLC-quantitative measurement of aflatoxin B1 and ochratoxin A residues, and molecular sequencing of aflR1 and pks genes. One hundred and twenty processed beef meat specimens (basterma, sausage, and minced meat; n = 40 for each) were collected from Ismailia Province, Egypt. Samples were prepared for total mold count, isolation, and identification of Aspergillus species. All samples were analyzed for the production of both Aflatoxin B1 and Ochratoxin A mycotoxins by HPLC. Molecular identification of Aspergillus flavus and Aspergillus ochraceus was performed using PCR amplification of the internal transcribed spacer (ITS) region; furthermore, the aflR1 and pks genes were sequenced. Results: The total mold count obtained from sausage samples was the highest one, followed by minced meat samples. The prevalence of A. flavus was (15%), (7.5%), and (10%), while the prevalence of A. ochraceus was (2.5%), (10%), and (0%) in the examined basterma, sausage, and minced meat samples, respectively. Using PCR, the ITS region was successfully amplified in all the tested A. flavus and A. ochraceus strains. Aflatoxin B1 was detected in six basterma samples (15%). Moreover, the ochratoxin A was detected only in four sausage samples (10%). The aflR1 and pks genes were amplified and sequenced successfully and deposited in the GenBank with accession numbers MF694264 and MF694264, respectively. Conclusions: To the best of our knowledge, this is the first report concerning the HPLC-Molecular-based approaches for the detection of aflatoxin B1 and ochratoxin A in processed beef meat in Egypt. The production of aflatoxin B1 and ochratoxin A in processed meat constitutes a public health threat. Aflatoxin B1 is commonly associated with basterma samples. Moreover, ochratoxin A was detected frequently in sausage samples. The routine inspection of mycotoxins in processed meat products is essential to protect human consumers. [ABSTRACT FROM AUTHOR]

Although Aspergillus flavus is a strong producer strain for kojic acid (KA), up till now there is no report on genes encoding KA. Conversely, KA genes from A. oryzae have been reported, with kojA, kojR and kojT genes, an enzyme gene, a transcription factor and a transporter, respectively, having been cloned and characterized. Thus, in the present study, we aimed to identify and characterize KA genes from A. flavus. To achieve this goal, we first produced by gamma irradiation A. flavus and A. oryzae mutants with improved levels of KA with respect to wild type (WT) strains. Then, we cloned orthologs of A. oryzae kojA, kojR and kojT from A. flavus. Finally, we analyzed the expression levels of KA genes in mutant (MT) and WT strains of both species. The mutants A. flavus HAk1-M2 and A. oryzae HAk2-M26 were found to produce KA at levels 2.5- and 3.02-fold higher than their parent strains after 7-10 days under shaking conditions. The cloned A. flavus genes AFLA_096040, AFLA_096050 and AFLA_096060, exhibited 99-100% similarity to kojA, kojR and kojT genes from A. oryzae, respectively. Gene expression analyses revealed that the expression levels of KA genes nicely overlap with KA levels both in A. flavus and A. oryzae, MT and WT strains. In addition, we cloned the laeA gene, which controls the biosynthesis of secondary metabolites from A. flavus. The results showed that, unlike A. oryzae, the expression of laeA in A. flavus does not correlate with KA biosynthesis. Our findings are of importance and reveal that the gene cluster comprising AFLA_096040, AFLA_096050 and AFLA_096060 is potentially involved in KA biosynthesis. [ABSTRACT FROM AUTHOR]

Background: Aflatoxins (AFs) are highly carcinogenic compounds produced by Aspergillus species in seeds with high lipid and protein contents. It has been known for over 30 years that peptone is not conducive for AF productions, although reasons for this remain unknown. Results: In this study, we showed that when Aspergillus flavus was grown in peptone-containing media, higher initial spore densities inhibited AF biosynthesis, but promoted mycelial growth; while in glucose-containing media, more AFs were produced when initial spore densities were increased. This phenomenon was also observed in other AF-producing strains including A. parasiticus and A. nomius. Higher peptone concentrations led to inhibited AF production, even in culture with a low spore density. High peptone concentrations did however promote mycelial growth. Spent medium experiments showed that the inhibited AF production in peptone media was regulated in a cell-autonomous manner. mRNA expression analyses showed that both regulatory and AF biosynthesis genes were repressed in mycelia cultured with high initial spore densities. Metabolomic studies revealed that, in addition to inhibited AF biosynthesis, mycelia grown in peptone media with a high initial spore density showed suppressed fatty acid biosynthesis, reduced tricarboxylic acid (TCA) cycle intermediates, and increased pentose phosphate pathway products. Additions of TCA cycle intermediates had no effect on AF biosynthesis, suggesting the inhibited AF biosynthesis was not caused by depleted TCA cycle intermediates. Conclusions: We here demonstrate that Aspergillus species grown in media with peptone as the sole carbon source are able to sense their own population densities and peptone concentrations to switch between rapid growth and AF production. This switching ability may offer Aspergillus species a competition advantage in natural ecosystems, producing AFs only when self-population is low and food is scarce. [ABSTRACT FROM AUTHOR]