REDUCTION OF FUNGI AND MYCOTOXINS FORMATION IN SEEDS BY GAMMA-RADIATION

Ninety samples of maize, chick-peas and groundnut seeds collected from the Egyptian market were found to be heavily contaminated by molds. Alternaria, Aspergillus, Cladosporium, Eurotium, Fusarium, Mucor, Penicillium and Rhizopus were the most common fungal genera isolated from nondisinfected seeds . Aspergillus alutaceus, A. flavus, Fusarium verticillioides and F. oxysporum were isolated from all surface-disinfected seeds and were reported to produce ochratoxin A, aflatoxin B₁ and zearalenone, respectively. Irradiation at a dose 4.0 kGy reduced the mold growth greatly relative to unirradiated controls. There was no growth at dose 5.0 kGy. On the basis of the radiation survival data, the decimal reduction values D₁₀ for A. alutaceus, A. flavus and F. verticilliodies were 0.70. 2.10 and 0.93 kGy in maize. A dose of 5 kGy inhibited the toxigenic molds and mycotoxin formation in seeds. Aflatoxin B₁ and ochratoxin A were detected in maize and chick-peas, whereas zearalenone was detected in maize samples. Application of radiation at a dose of 6.0 kGy detoxified aflatoxin B₁ by 74.3–76.7%, ochratoxin A by 51.3–96.2% and zearalenone by about 78%.     

INFLUENCE OF MOISTURE CONTENT AND STORAGE TEMPERATURE ON THE PRODUCTION OF AFLATOXIN BY ASPERGILLUS FLAVUS EA-81 IN MAIZE AFTER EXPOSURE TO GAMMA RADIATION

The effect of gamma radiation on aflatoxin production by Aspergillus flavus EA-81 in maize with different initial moisture levels was determined over a 15-day period. The viability of A. flavus on maize decreased over time with increasing moisture contents and storage at 8C. After 45 days at 28C, levels of viable conidiospores of A. flavus increased from 4.5 × 10⁷ to about 3.0 × 10⁸ per gram of maize. Levels of aflatoxin B₁ produced by A. flavus were 10 μg kg^-1 in the maize stored at 8C after 45 days. Production of aflatoxin was highest at 40% moisture and 28C. Irradiation of 1.0 or 2.0 kGy greatly reduced the level of mold growth relative to unirradiated controls. A dose of 4.0 kGy eliminated all viable fungi. Aflatoxin B₁ production decreased with increased levels of irradiation and was negligible at 4.0 kGy. When maize was inoculated after irradiation and stored, the spore counts and aflatoxin levels were higher than in unirradiated and inoculated controls after 30 days. Apparently, the natural competitive microflora prevented growth and thus limited higher concentrations of aflatoxin in maize.     

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 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.     

EFFECT OF GAMMA IRRADIATION ON AFLATOXIN B1 PRODUCTION BY ASPERGILLUS FLAVUS IN GROUND BEEF STORED AT 5C

The effect of γ-irradiation for controlling the production of aflatoxin B₁ by Aspergillus flavus in ground beef stored at 5C for 2 weeks was investigated. Aspergillus, Penicillium, Cladosporium, Mucor, Scopulariopsis, Candida and Rhodotorula were the most common fungal genera contaminating ground beef. A. flavus and A. niger were the most common Aspergillus spp. Aspergillus flavus isolates were able to produce aflatoxin B₁ in ground beef. Only 3 (20%) samples of ground beef were contaminated with aflatoxin B₁ (25–45 μg/Kg). Gamma irradiation dose levels resulted in an immediate reduction in the total numbers of A. flavus. No growth or aflatoxin B₁ production occurred at 1.50 kGy during storage.     

Influence of competitive microbial flora and inoculum size of Penicillium purpurogenum on the production of rubratoxin-B in corn medium before and after exposure to gamma radiation

The influence of inoculum size on the production of rubratoxin-B by P. purpurogenum was estimated on unirradiated and irradiated corn stored for prolonged periods at 28 °C and at a moisture content of 25%. Irradiation at doses of 1.0–3.0 kGy reduced the level of mould growth greatly relative to unirradiated controls. Rubratoxin-B production in corn grains at all doses after 40 days was less than the unirradiated control level and was below the detection limit above 4.0 kGy. Non-sterile corn that was inoculated with conidia of the fungus and then irradiated with 2.0 kGy gamma rays, produced lower rubratoxin-B levels compared with unirradiated controls. When corn was inoculated with 10⁵ spores per gram after irradiation, the spore count and rubratoxin-B level were higher than the unirradiated control after 60 days. The total viable counts of endogenous fungal flora in the corn were reduced by the 2-kGy radiation dose, but after 20 days of incubation the numbers of P. purpurogenum increased with greater enhancement of rubratoxin-B production.     

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.     

Effects of E‐beam irradiation and vacuum packaging on biogenic amines formation in common carp (Cyprinus carpio) fillets

The effects of vacuum packaging followed by E‐beam irradiation treatment on the shelf life of common carp (Cyprinus carpio) fillets were studied by measuring biogenic amines and sensory analysis. Samples were irradiated at doses of 0.10, 0.50, 1.0 and 2.0 kGy. Putrescine, cadaverine, histamine and tyramine showed very good correspondence with the irradiation dose and the time of storage. Spermine, spermidine, tryptamine and phenylethylamine did not show statistically significant changes with the time of storage. According to the sensory assessment and biogenic amines index (BAI), the shelf life of unirradiated common carp fillets was found to be approximately seven days. The 1 and 2 kGy irradiation doses extended the shelf life of samples up to 66 and 77 days, respectively. High values of correlation coefficients (r < ?0.86) between BAI and sensory evaluation indicated that BAI could be considered as a quality indicator of common carp fillets.     

MICROBIOLOGICAL AND CHEMICAL PROPERTIES OF IRRADIATED MANGO

Fourteen fungal species belonging to five genera , Aspergillus, Cladosporium, Penicillium, Fusarium and Scoplariopsis were isolated from the surface of mango fruits , (Mangifera indica L.) variety "Zebda". Mycotoxin production by isolated molds was tested in Czapek-Dox broth as well as mango pulp. The irradiation decimal reduction doses (D₁₀ values) of the molds were determined in either physiological saline solution or mango pulp. The effects of ionizing radiation in combination with hot water-dipping (55C/5 min) on shelf-life extension and the microbiological, chemical, rheological and organoleptic properties of mango fruit (at mature green stage) stored at 12°C and 80–85% relative humidity were determined. Results showed that Zebda mango irradiated with doses of 1.0 kGy can be stored 50 days at 12°C without any undesirable changes to measured nutrient and sensory quality when compared to unirradiated mangoes refrigerated for 25 days.     

Use of PCR for Detecting Aspergillus flavus in Maize Treated by Gamma Radiation Process

The aim of this study was to verify the effects of gamma radiation process on the fungal DNA and the application of PCR in the detection of Aspergillus flavus in irradiated maize grains. The samples were inoculated with a toxigenic strain and incubated under controlled conditions of relative humidity, water activity, and temperature for 15 days. After incubation, the samples were treated with gamma radiation with doses of 5 and 10 kGy and individually analyzed. The use of PCR technique showed the presence of DNA bands of Aspergillus flavus in all irradiated samples that showed no fungal growth in agar medium.     

Lipid oxidation of irradiated pork products

The effect of irradiation (2, 5 and 8 kGy) and vacuum storage for 60 days on fatty acid and cholesterol oxidation was determined in three Italian cured pork products (salame Milano, coppa and pancetta).A significant increase in the degree of fatty acid oxidation was observed starting from 8 kGy irradiation dose. Unlike the unirradiated samples, the vacuum storage was not sufficient to curb fatty acid oxidation in the irradiated pork products.The cholesterol oxidation of all of the pork products investigated was affected by the highest dose of irradiation and sometimes by storage. The effect induced by the ionising radiation was different depending on the type of pork product. The cholesterol oxide molecules were qualitatively similar in both irradiated and unirradiated pork products and the levels detected were about 100 times lower than the toxic level for in vitro and in vivo experiments.     

Influence of γ‐irradiation and maize lipids on the production of aflatoxin B1 by Aspergillus flavus

The effect of γ‐irradiation and maize lipids on aflatoxin B₁ production by Aspergillus flavus artificially inoculated into sterilized maize at reduced water activity (a_w 0.84) was investigated. By increasing the irradiation doses the total viable population of A. flavus decreased and the fungus was completely inhibited at 3.0 kGy. The amounts of aflatoxin B₁ were enhanced at irradiation dose levels 1.0 and 1.5 kGy in both full‐fat maize (FM) and defatted maize (DM) media and no aflatoxin B₁ production at 3.0 kGy γ‐irradiation over 45 days of storage was observed. The level in free lipids of FM decreased gradually, whereas free fatty acid values and fungal lipase activity increased markedly by increasing the storage periods. The free fatty acid values decreased by increasing the irradiation dose levels and there was a significant enhancement of fungal lipase activity at doses of 1.0 and 1.50 kGy. The ability of A. flavus to grow at a_w 0.84 and produce aflatoxin B₁ is related to the lipid composition of maize. The enhancement of aflatoxin B₁ at low doses was correlated to the enhancement of fungal lipase activity.     

INCIDENCE OF MYCOTOXIC MOLDS IN DOMESTIC AND IMPORTED CHEESES12

Domestic and imported cheeses were studied to determine the incidence of mycotoxin producing molds. The total incidence of molds in visibly non-moldy cheese was very low. Isolation of molds from plate counts, and directly from samples, showed that the major portion of the flora was made up of Penicillium species; 86% in domestic cheeses and 80% in imported cheeses. Many of the Penicillium isolates were capable of growing at low storage temperatures. Mold counts done at 5°C, and prolonged storage of cheese samples at 5°C indicated a potential for considerable mold growth on cheese during refrigerated storage. While the overall incidence of known mycotoxin producing molds was low, a number of potentialy toxic species were found, including P. cyclopium, P. viridicatum, A. flavus and A. ochraceus. These species accounted for 4.4% of all the isolates from domestic cheeses and 4.0% from imported cheeses. Screening of all mold isolates for production of several known mycotoxins showed that a number of isolates (14.1% of all molds in domestic cheeses and 11.5% in imported cheeses) were capable of producing certain mycotoxins including patulin, penicillic acid, ochratoxin A, citrinin and aflatoxins.     

Investigation on the toxicity of fungi from rootstock snacks

Chicken embryo bioassay was used to monitor the toxicity of extracts from rootstock snack samples during a 210-day storage period. Results show that the relative toxicity values which were initially very low increased significantly as from 120th day (when 32% mortality was recorded) up till the last day when 73% was obtained. Toxicity of extracts from axenic cultures of 12 fungal species isolated from the snack samples was also determined. The strains of Aspergillus chevalieri, Rhizopus nigricans and Rhizopus sp investigated were nontoxic. A. niger, A. flavus, A.fumigatus, Penicillium chrysogenum, A. parasiticus, P. citrinum, A. ochraceus, Fusarium monifilorme and A. candidus were found to be toxic in decreasing order of potency as recorded after 30 days of growth at room temperature on substrate of 0.90 water activity level. Trends in the moisture content levels, pH and the incidence of fungal growth on the snack samples were also studied. Results suggest that storage for a period not exceeding 90 days and maintenance of safe moisture level would control mould growth and the associated mycotoxins in the snack.     

Fungal mycoflora and mycotoxins in Korean polished rice destined for humans

Rice samples collected from the Republic of Korea were analyzed for fungal mycoflora and mycotoxins: fumonisins, ochratoxin A, trichothecenes, and zearalenone. The potential of the fungi to produce each mycotoxin was also examined, so that the fungal isolates associated with mycotoxins occurring in rice could be verified. Penicillium citrinum and Aspergillus candidus were the most prevalent species infecting the samples, while Fusarium proliferatum was found as the dominant Fusarium species. Ochratoxin A was the most commonly detected mycotoxin analyzed in the present study; moreover, its level in some samples was above the EU tolerable limit (3 ng/g). According to rice culture experiments, it was revealed that in Korea, fumonisins detected in rice were due to F. proliferatum infection, whereas the occurrence of ochratoxin A was caused by Penicillium verrucosum, though there were no symptoms of disease in rice found in any sample. Furthermore, there appears to be an uneven geographical distribution of P. verrucosum as well as ochratoxin A in that most of them are found in the rice samples produced in the northern region of Korea.     

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.     

Public Health Significance of Molds and Mycotoxins in Fermented Dairy Products

Mold growth on cheese and other fermented dairy products is a common and recurring problem. Potential mycotoxin contamination is serious since some molds can grow and produce mycotoxins at temperatures as low as ?2 to 10°C. Work can be divided into: 1) incidence, types, and mycotoxin-producing potential of molds in fermented dairy products, 2) experimental mycotoxin production on cheese under conditions of storage and aging of cheese, 3) natural occurrence of mycotoxins in commercial samples of cheese, and 4) potential toxicity of Penicillium roqueforti and its significance in blue veined cheeses.Molds most common on cheese and fermented dairy products are Penicillium species. Mycotoxins produced by these organisms are penicillic acid, patulin, ochratoxin A, and citrinin. Percentages of molds in cheese capable of producing some commonly studied mycotoxins ranged from 1.8% to 12.4%. Cheese is an excellent substrate for mold growth but a poor substrate for mycotoxin production. Several natural occurrences of mycotoxins in cheese include small and variable amounts of patulin, penicillic acid, sterigmatocystin (600 µg/kg), penitrem A, and mycophenolic acid. Penicillium roqueforti is capable of producing toxic alkaloids and other compounds. The significance of these substances for human health is unclear.The decision to trim or to discard moldy cheese can be aided by considering the risk versus benefit based on storage history (temperature), extent of mold growth, appearance of mold (color), and size of cheese.     

Mycotoxins in corn and wheat silage in Israel

Silage is an important feed source for intensive dairy herds worldwide. Fungal growth and mycotoxin production before and during silage storage is a well-known phenomenon, resulting in reduced nutritional value and a possible risk factor for animal health. With this in mind, a survey was conducted to determine for the first time the occurrence of mycotoxins in corn and wheat silage in Israel. A total of 30 corn and wheat silage samples were collected from many sources and analysed using a multi-mycotoxin method based on LC-MS/MS. Most mycotoxins recorded in the present study have not been reported before in Israel. Overall, 23 mycotoxins were found in corn silage; while wheat silage showed a similar pattern of mycotoxin occurrence comprising 20 mycotoxins. The most common post-harvest mycotoxins produced by the Penicillium roqueforti complex were not found in any tested samples, indicative of high-quality preparation and use of silage. Moreover, none of the European Union-regulated mycotoxins – aflatoxin B1, ochratoxin, T-2 toxin, diacetoxyscirpenol and deoxynivalenol – were found above their limits of detection (LODs). The Alternaria mycotoxins – macrosporin, tentoxin and alternariol methyl ether – were highly prevalent in both corn and wheat silage (>80%), but at low concentrations. The most prominent (>80%) Fusarium mycotoxins in corn silage were fusaric acid, fumonisins, beauvericin, monilifomin, equisetin, zearalenone and enniatins, whereas in wheat silage only beauvericin, zearalenone and enniatins occurred in more than 80% of the samples. The high prevalence and concentration of fusaric acid (mean = 765 µg kg^–1) in Israeli corn silage indicates that this may be the toxin of highest potential concern to dairy cow performance. However, more data from different harvest years and seasons are needed in order to establish a more precise evaluation of the mycotoxin burden in Israeli silage.     

The effectiveness of electron beam irradiation to reduce or eliminate mould in cork stoppers

The objective of this research project was to reduce or eliminate moulds known to cause TCA using electron beam (e‐beam) irradiation. Four types of cork were inoculated with Paecilomyces viridis, Penicillium glabrum, Penicillium chrysogenum, Mucor racemosus, Trichoderma viride, Aspergillus oryzae and Cladosporium oxysporum. Cork samples were treated with 5, 10 or 15 kilograys (kGy) of e‐beam irradiation, respectively. Sensory evaluation revealed that irradiation had little or no effect on the overall quality of wine with irradiated corks stored for 56 months. Cork samples viewed under a scanning electron microscope (SEM) showed some structural changes compared to control corks. The results suggest that e‐beam irradiation can significantly reduce or eliminate the moulds known to cause TCA in cork.     

Effect of gamma-irradiation on the natural occurrence of Fusarium mycotoxins in wheat,flour and bread

A survey was carried out to obtain data on the occurrence of Fusarium mycotoxin in wheat and flour samples collected from local markets in Egypt and to study the influence of gamma-irradiation on controlling the occurrence of these mycotoxins in wheat, flour and bread. Deoxynivalenol (DON) was detected in five samples of wheat at levels ranging from 103 to 287 μg/kg and one sample each of flour and bread at concentrations 188 and 170 μg/kg. Zearalenone (ZEN) was detected in ten samples of wheat at levels from 28 to 42 μg/kg and four samples each of flour and bread at concentrations of 95 and 34 μg/kg, respectively. T-2 toxin was detected only in one sample each of wheat, flour and bread at concentrations of 2.9, 2.2 and 2.3 μg/kg, respectively. Gamma-irradiation at dose level of 6 kGy completely eliminated fungal flora in flour and wheat. DON, ZEN and T-2 toxin concentrations are reduced to 85, 20 and 2.0 μg/kg for wheat and to 125, 45, and 1.0 μg/kg for flour after 4 kGy exposure and a sharp drop in Fusarium toxin levels occurred at 6 kGy and was eliminated at 8 kGy. Bread prepared from 6 kGy was contaminated with Fusarium toxin at levels below 5 μg/kg. It was noticed that gamma-irradiation reduce greatly the natural occurrence of Fusarium mycotoxins in bread.