Effect of gamma irradiation and heat treatment on the artificial contamination of maize grains by Aspergillus flavus Link NRRL 5906

Maize (Zea mays L.) is one of the main crops, which is easily susceptable to Aspergillus flavus infection resulting in huge losses worldwide. This study was carried out to investigate the effect of combining heat and irradiation treatments in controlling the fungal growth in maize grains. Surface disinfected maize grains were artificially contaminated with spores of Aspergillus flavus Link NRRL 5906, and then exposed to gamma radiation with doses of 3.0, 4.0 and 5.0 kGy. The samples were additionally heat treated at 60 °C for 30 min. The heat and irradiation treatments showed a synergistic effect on controlling Aspergillus flavus growth. The heat treatment reduced the required radiation dose of about 0.5–1.0 kGy when 4.0 kGy or 5.0 kGy irradiation was used. The combined heat and irradiation treatment of moisture reduced the average CFU by 8 log cycles when 4 kGy or 5 kGy irradiation was used and by 7 log cycles when 3 kGy irradiation was used. The heat treatment of moisture alone reduced the average CFU by only by 0.8 log cycles. Combining irradiation with heat treatment to reduce the required radiation dose is very useful especially when there is a concern over biological side effects of irradiation.     

Production of aflatoxins in sausage,salami, sucuk and kavurma

Forty nine meat product samples were examined for the fungal genera. Penicillium sp. was detected in 74.8% of samples. No sample contained Aspergillus parasiticus or Aspergillus flavus. Production of aflatoxins in sausage, salami, sucuk and kavurma by A. parasiticus and A. flavus was studied at different temperatures. A. parasiticus and A. flavus produced no aflatoxins on meat products samples at 15°C. Sucuk was a poor substrate for A. parasiticus and A. flavus at 25°C. Sausage, salami and kavurma were favorable substrates for aflatoxin production by A. parasiticus at 25°C.     

Mold and Aflatoxin Reduction by Gamma Radiation of Packed Hot Peppers and Their Evolution during Storage

The effect of gamma radiation on moisture content, total mold counts, Aspergillus counts, and aflatoxins of three hot pepper hybrids (Sky Red, Maha, and Wonder King) was investigated. Whole dried peppers packed in polyethylene bags were gamma irradiated at 0 (control), 2, 4, and 6 kGy and stored at 25°C for 90 days. Gamma radiation proved to be effective in reducing total mold and Aspergillus counts in a dose-dependent relationship. Total mold counts in irradiated peppers immediately after treatments were significantly lowered compared with those in nonirradiated samples, achieving 90 and 99% reduction at 2- and 4-kGy doses, respectively. Aspergillus counts were significantly reduced, by 93 and 97%, immediately after irradiation at doses of 2 and 4 kGy, respectively. A radiation dose of 6 kGy completely eliminated the population of total molds and Aspergillus fungi. The evolution of total molds in control and irradiated samples indicated no further fungal proliferation during 3 months of storage at 25°C. Aflatoxin levels were slightly affected by radiation doses of 2 and 4 kGy and showed a nonsignificant reduction of 6% at the highest radiation dose of 6 kGy. The distinct effectiveness of gamma radiation in molds and aflatoxins can be explained by the target theory of food irradiation, which states that the likelihood of a microorganism or a molecule being inactivated by gamma rays increases as its size increases.     

Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR

Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10⁶ spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies.     

Specific detection of Aspergillus parasiticus in wheat flour using a highly sensitive PCR assay

Aspergillus parasiticus is one of the most important aflatoxin-producing species that contaminates foodstuffs and beverages for human consumption. In this work, a specific and highly sensitive PCR protocol was developed to detect A. parasiticus using primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). The assay proved to be highly specific for A. parasiticus when tested on a wide range of related and other fungal species commonly found in commodities, and allowing discrimination from the closely related A. flavus. Accuracy of detection and quantification by conventional PCR were tested with genomic DNA obtained from wheat flour artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10⁶ spore/g could be detected by the assay directly without prior incubation of the samples. The assay described is suitable for incorporation in routine analyses at critical points of the food chain within HACCP strategies.     

Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour

Aspergillus flavus is frequently found in food, producing a wide variety of toxins, aflatoxins being the most relevant in food safety. A specific PCR-based protocol for this species is described which allowed discrimination from other closely related species having different profiles of secondary metabolites from the Aspergillus Section Flavi, particularly A. parasiticus. The specific primers were designed on the multi-copy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA) and were tested in a wide sample of related species and other fungal species commonly found in food. The PCR assay was coupled with a fungal enrichment and a DNA extraction method for wheat flour to enhance the sensitivity of the diagnostic protocol. The results indicated that the critical PCR amplification product was clearly observed for wheat flour contaminated by 10² spores after 16 h of incubation.     

CONTAMINATION OF GRAINS BY MYCOTOXIN-PRODUCING MOLDS AND MYCOTOXINS AND CONTROL BY GAMMA IRRADIATION

Ninety random grain samples were collected and analyzed for mycotoxins, and the effect of gamma irradiation on the production of mycotoxins in grains was studied. Aspergillus, Penicillium, Mucor, Rhizopus, Fusarium, Alternaria, Scopulariopsis and Cladosporium were the most common fungal genera isolated from grains. Aspergillus flavus, Aspergillus niger, Aspergillus candidus, Aspergillus ochraceus, Penicillium citrinum, Penicillium expansum, Penicillium citreonigrum, Penicillium purpurogenum, Penicillium griseofulvum and Penicillium verrucosumwere the most common Aspergillus and Penicillium species in grains. Out of 120 Aspergillus and Penicillium isolates, 80 were mycotoxin producers. Analysis of grains revealed the occurrence of aflatoxin B₁ ochratoxin A, cycolopiazonic acid and citrinin. Of the 90 samples, 67 were positive for one or more mycotoxin. Irradiation of grains at dose of 2.0 and 4.0 kGy decreased significantly the total fungal counts compared with unirradiated controls. After 100 days of storage at room temperature, the unirradiated grains were contaminated with high concentrations of mycotoxins as compared with irradiated 4.0‐kGy samples. Mycotoxin production in grains decreased with increasing irradiation doses and was not detected at 6.0 kGy over 100 days of storage.     

Mycobiota of cocoa: from farm to chocolate

The present work was carried out to study the mycobiota of cocoa beans from farm to chocolate. Four hundred and ninety-four samples were analyzed at various stages of cocoa processing: (i) primary stage at the farm (fermentation, drying, and storage), (ii) secondary stage at processing (testa, nibs, liquor, butter, cake and powder) and (iii) the final chocolate product (dark, milk, white and powdered) collected from retail outlets. Direct plating or dilution plating on Dichloran 18% Glycerol agar were used for cocoa beans and processed product analyses, respectively. Fungi were isolated and identified using different keys of identification. The largest numbers and diversity of fungi were observed in the samples collected at the farm, especially during drying and storage. The species with the highest occurrence among samples were: Absidia corymbifera, Aspergillus sp. nov., A. flavus, Penicillium paneum and yeasts. A total of 1132 potentially toxigenic fungi were isolated from the following species or species groups: A. flavus, Aspergillus parasiticus, Aspergillus nomius, Aspergillus niger group, Aspergillus carbonarius and Aspergillus ochraceus group. The highest percentage of toxigenic fungi was found at the drying and storage stages. The industrial processing reduced the fungal contamination in all fractions and no fungi were found in the final chocolate products. The knowledge of which fungi are dominant at each processing stage of cocoa provides important data about their ecology. This understanding leads to a reduction in fungal spoilage and mycotoxin production in this product.     

Contamination with storage fungi of human food from Cameroon

In a mycological study, a total of 95 human food samples were investigated to evaluate the incidence of fungal contamination in Cameroon by conventional identification method and partly confirmed by DNA sequencing. The isolated fungal spp. were further studied to determine their toxigenic potentials. The investigation revealed the predominance of Aspergillus and Penicillium with 96% of samples contaminated with at least one species of these fungi, whereas the incidence of co-contamination of samples was 85%. Aspergillus flavus and Aspergillus parasiticus (Flavi section) were the most predominant species contaminating mainly maize and peanuts. In addition, P. crustosum and P. polonicum were the most common contaminants belonging to the genus Penicillium. On the other hand, A. ochraceus (Circumdati section) registered a low incidence rate of 5%, including other members of the Aspergillus group. Other members of the genera Rhizopus and Alternaria spp. were also registered in the study. A majority of fungal strains of A. ochraceus, A. parasiticus, P. crustosum and P. polonicum isolated were toxigenic, producing the mycotoxins tested for, while none was detected in cultures of A. fumigatus. The high incidence rate of fungi contamination coupled with their potentials in producing mycotoxins gives a strong indication that the samples tested may likely be contaminated with various mycotoxins. There is need for further study to assess the incidence of mycotoxins contamination in similar food samples.     

Contamination of freshly harvested Bambara groundnut (Vigna subterranea) seed from Mpumalanga,South Africa,with mycotoxigenic fungi

Freshly harvested Bambara groundnut (BGN) is occasionally consumed raw and can potentially become infected with mycotoxingenic field fungi. In this study, BGN samples were obtained from 12 farms in three districts of Mpumalanga in South Africa. Eight pooled samples were screened for multi-mycotoxin contamination using Ultra Performance Liquid-Chromatography tandem mass spectrometry (UPLC-MS/MS). To identify mycoflora, 12 samples were screened using conventional and molecular methods. Selected potential mycotoxin producing isolates were screened for mycotoxins using UPLC-MS/MS. No mycotoxins were detected on the freshly harvested BGN samples, but they were infected with various mycotoxin producing fungal species namely Aspergillus flavus (50%), Penicillium citrinum (25%), Penicillium oxalicum (17%), Penicillium citreoviridin (0.8%), and Fusarium verticillioides (0.8%). Following screening of selected fungal cultures, aflatoxin B₁ (0.4, 0.45 and 0.4 ppm) and fumonisin B₁ (0.7 ppm) were detected from A. flavus and F. verticillioides, respectively. Identification of mycotoxigenic fungi on freshly harvested BGN presents a potential health risk.     

Effect of γ‐irradiation on aflatoxin B1 production by Aspergillus flavus and chemical composition of three crop seeds

The effect of γ‐irradiation on aflatoxin B₁ production by Aspergillus flavus, and the chemical composition of some different crop seeds were investigated. A. flavus infected seeds behaved differently according to their principal constituents. A. flavus caused an increase in protein and decrease in lipids and carbohydrate contents of wheat, soyabean and fababean seeds. Growth of A. flavus and production of aflatoxin B₁ was inhibited at a dose level of 5 kGy. A. flavus utilizes carbohydrates of seeds for its growth and aflatoxin production. Crops were arranged, in descending order, according to aflatoxin produced in seeds as wheat > soyabean > fababean. There were no changes in chemical constituents of irradiated seeds, such as protein, lipids, and carbohydrates.     

Isolation, enumeration and PCR characterization of aflatoxigenic fungi from food and feed samples in India

A total of 54 market samples comprising nine different food and feed commodities from Mysore city were examined in order to isolate aflatoxin-producing fungi as well as to assess aflatoxins in the commodities. Thirty-two samples were contaminated with aflatoxigenic fungi and the total mycoflora and aflatoxigenic fungi in different food and feed commodities were in the range of 0.2–260 and 0–100 cfu×10³/g, respectively. In total, 136 fungi were isolated, of which 32 were Aspergillus flavus strains and 26 of them were found to produce aflatoxins. A. flavus group of fungi comprising A. flavus, A. parasiticus, A. oryzae, A. sojae were characterized by using Aspergillus differential medium and PCR. The PCR was performed using two different sets of primers specifically targeted to aflR and omt genes of aflatoxin biosynthesis pathway. Most of the fungi belonging to A. flavus group reacted positively with the primers resulting in expected size amplicons of 796 bp for aflR and 404 bp for omt. Among the nine commodities screened for aflatoxin only, groundnut and groundnut cake were contaminated with aflatoxins B₁ and B₂. The aflatoxin contamination in these commodities exceeded the Indian regulatory limit of 30 μg/kg.     

A mycological survey on mixed poultry feeds and mixed rabbit feeds

The mycoflora of 55 samples of mixed poultry feed and mixed rabbit feed were studied. Differences in the mycoflora between the two kinds of feed were studied. Fungal counts ranged from < 10 ² to 10 ⁵ cfu g^?1, the lowest counts being in the samples of rabbit feeds. Seventy-three fungal species belonging to 28 genera were identified. The predominant species were Aspergillus flavus, A candidus, A amstelodami, Penicillium chrysogenum and Fusarium moniliforme. Aphanocladium album, ranking sixth in the samples overall, showed exceptional incidence compared with published data. Fusarium moniliforme, A flavus and A amstelodami had the highest individual percentage counts. The F. moniliforme and A candidus percentage counts were significantly higher in mixed poultry feeds than in mixed rabbit feeds, quite the reverse of the A amstelodami and P chrysogenum percentage counts.     

A field survey of pistachio (Pistacia vera) nut production and storage in Turkey with particular reference to aflatoxin contamination.

A field study of soils and pistachio nuts in three major pistachio-growing areas of Turkey has been undertaken. The predominant soil fungus isolated from all three areas was Aspergillus niger; isolates of Fusarium, Trichoderma, Mucor and Rhizopus species were also common. A flavus and A. ochraceus and related organisms were isolated only from the plateau of Gaziantep. The dominant external mycoflora of the immature nuts from all sites consisted of A. niger, A. flavus and Penicillium spp. There was no evidence for fungal contamination of immature endosperms nor of freshly dehulled nuts, but stored dehulled nuts and dust samples taken from ware-houses were extensively contaminated with A. flavus, A. niger and A. ochraceus. Of 43 A. flavus isolates tested, 20 produced aflatoxins on laboratory media or on rice; all 11 isolates of A. ochraceus tested produced ochratoxin A on laboratory media. The results obtained are discussed in relation to agricultural and marketing practices for Turkish pistachio nuts.     

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.     

Differentiation of toxigenic fungi using hyperspectral imagery

Some pathogenic fungi, Aspergillus flavus for example, produce mycotoxins that can contaminate grain products including wheat and corn. The contaminated grain poses a threat to the health of both humans and animals. Therefore, from the perspective of food safety and protection, it is important to detect and identify the different toxin-producing fungi encountered in food production. Earlier studies examined various spectral-based, non-destructive methods for the detection of fungi and toxins. The present report focused on the feasibility of using spectral image data for fungal species classification. A tabletop hyperspectral imaging system, VNIR-100E, was used for spectral and spatial data acquisition. A total of five fungal species were selected for a two-part experiment: Penicillium chrysogenum, Fusarium moniliforme (verticillioides), Aspergillus parasiticus, Trichoderma viride, and Aspergillus flavus. All fungal isolates were cultured on media under laboratory conditions and were imaged on day 5 of growth. The objective of the study was to use visible near-infrared hyperspectral imagery to differentiate fungal species. Results indicate that all five fungi are highly separable with classification accuracy of 97.7%. In addition, all five fungi could be classified by using only three narrow bands (bandwidth = 2.43 nm) centered at 743 nm, 458 nm, and 541 nm.     

Production of fungal enzymes and proteins from high salt cheese wheys

A fermentation procedure using wheys from buffalo milk, sweet rennet and salted wheys containing up to 15% NaC1, and based on the selection of salt-tolerant fungal cultures for production of microbial proteins and some economically important enzymes, is described. Certain strains of Aspergillus flavus, A. niger, A. ochraceus and A. terreus gave yields of mycelium ranging between 1.5 and 30 g on a dry weight basis. Strong proteolytic activities were exhibited by A. ochraceus, A. flavus and, to a lesser extent, by A. niger in the presence of salt up to 10% as final concentration. Powerful milk-clotting activities were also detected in culture supernatants of A. flavus and A. ochraceus. Extracellular β-galactosidases were released in to the culture fluids of these fungal strains with variable activities, depending upon the salt content of the growth medium. The application of the procedure is discussed.     

Effect of synthetic antioxidants on stored maize grain mycoflora in situ

BACKGROUND: The influence of a mixture of butylated hydroxyanisole (BHA) and propyl paraben (PP) (each at a concentration of 20 mmol L^?1) on mycoflora and Aspergillus section Flavi populations in stored maize grain was evaluated. A survey of 120 maize samples was carried out from June to November 2005. RESULTS: The predominant populations in non‐treated (control) maize between the first and sixth sampling periods were Aspergillus section Flavi and Penicillium. Aspergillus flavus was the fungus most frequently isolated from both control and antioxidant‐treated kernels. All samples of control and antioxidant‐treated maize kernels were negative for aflatoxins during the 6 month storage period. Aspergillus flavus and A. parasiticus strains showed a variable ability to produce aflatoxins. The contribution of the strains to silo community toxigenicity was higher for A. flavus L (large) and S (small) strains in the fourth sampling period. CONCLUSION: Antioxidant treatment negatively affected natural maize mycoflora and Aspergillus section Flavi populations between the second and sixth months of storage. Copyright © 2007 Society of Chemical Industry     

Multiplex Real‐Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera

Abstract: Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real‐time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene‐producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON‐ and OTA‐producing fungi can be detected and quantified in a single reaction tube using this multiplex real‐time PCR method. Practical Application: This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal‐based foods.     

Mycoflora and deoxynivalenol in whole wheat grains (Triticum aestivum L.) from Southern Brazil

The fungal species Fusarium graminearum is related to deoxynivalenol (DON) formation. The aim of this study was to evaluate mycoflora and DON occurrence in 53 whole wheat grain samples collected in Southern Brazil during the 2012 crop. Wheat grains showed adequate values of water activity ranging from 0.48 to 0.72, within the required limits of moisture content, ranging from 9.1% to 13.9%. In addition, low counts of fungal colonies, ranging from 10 to 8.2 × 10², were found. For Fusarium genera, there was predominance of Fusarium verticillioides (34%) and F graminearum (30.2%). For Aspergillus species, 37.7% of Aspergillus flavus was determined. Regarding the Penicillium species, Penicillium digitatum (49%) was the most found species. DON was detected in 47.2% (25 out of 53) of the samples analysed, with levels ranging from 243.7 to 2281.3 µg kg^?1 (mean: 641.9 µg kg^?1).