Studies on the ability of mycotoxins to diffuse in bread

Summary The moulds Aspergillus parasiticus (aflatoxins B₁, B₂, G₁, G₂, and M₁), A. ochraceus (ochratoxin A) and Penicillium chrysogenum (citrinin) were grown on whole wheat bread either in the presence or absence of oxygen. In the presence of oxygen, both A. parasiticus and A. ochraceus developed dense colonies and formed considerable amounts of mycotoxins whereas Penicillium chrysogenum only grew and produced citrinin on the surface of the bread. In the absence of oxygen fungal growth did not occur and most of the toxins were undetectable even in regions of bread immediately adjacent to the moulds although a very slight diffusion of the aflatoxins B₁ and G₁ through 1 cm was observed. It is concluded that diffusion of the tested mycotoxins from hyphae into bread is not a problem for food safety.     

Mycotoxins as human carcinogens—the <Emphasis Type="Italic">IARC Monographs</Emphasis> classification

Humans are constantly exposed to mycotoxins (e.g. aflatoxins, ochratoxins), mainly via food intake of plant and animal origin. The health risks stemming from mycotoxins may result from their toxicity, in particular their carcinogenicity. In order to prevent these risks, the International Agency for Research on Cancer (IARC) in Lyon (France)—through its IARC Monographs programme—has performed the carcinogenic hazard assessment of some mycotoxins in humans, on the basis of epidemiological data, studies of cancer in experimental animals and mechanistic studies. The present article summarizes the carcinogenic hazard assessments of those mycotoxins, especially aflatoxins (aflatoxin B₁, B₂, G₁, G₂ and M₁), fumonisins (fumonisin B₁ and B₂) and ochratoxin A (OTA). New information regarding the genotoxicity of OTA (formation of OTA-DNA adducts), the role of OTA in oxidative stress and the identification of epigenetic factors involved in OTA carcinogenesis–should they indeed provide strong evidence that OTA carcinogenicity is mediated by a mechanism that also operates in humans–could lead to the reclassification of OTA.     

Mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds for human consumption

A survey was carried out to obtain data on the occurrence of mycotoxins and the mycotoxin-producing potential of fungi isolated from nuts (almonds, peanuts, hazelnuts, pistachio nuts) and sunflower seeds in Spain. Thin-layer chromatography was used to separate the toxins. Aflatoxins were detected in one sample of almonds (95 ppb aflatoxin B₁ and 15 ppb aflaxtoxin B₂) and in one sample of peanuts at a level below 10 ppb of aflatoxin B1. 100% of samples showed variable incidence of fungal contamination. The predominant fungi present in samples were Penicillium spp, Aspergillus niger, A. flavus, A. glaucus and Rhizopus spp. The results showed that isolates of different species were able to produce aflatoxins B₁, B₂, G₁, and G₂, sterigmatocystin, ochratoxin A, patulin, citrinin, penicillic acid, zearalenone, and griseofulvin.     

Survey of aflatoxins and ochratoxin A in stored tubers ofCyperus esculentus L.

The mold incidence, moisture contents, pH and levels of mycotoxins (aflatoxins B₁, G₁ and ochratoxin A) on/of/in rootstock snack (tubers ofCyperus esculentus L.) samples were monitored during a 150-day storage period. Whereas the mold incidence, moisture and mycotoxin levels increased with storage time, the pH declined during the same period. Altogether, 12 fungal species, mostly toxigenic, includingAspergillus flavus, A. parasiticus andA. ochraceus were isolated. At collection period only 3 of the 9 snack samples analysed contained trace amounts of aflatoxins. By 120th day, all the 9 samples were contaminated and the average levels were 454 and 80 ppb for aflatoxin B₁ and aflatoxin G₁ respectively on the 150th day. Ochratoxin A was not detected before 120th day and then only at low levels, occuring in a maximum of four samples and ranging between 10 and 80 ppb.     

Mycotoxins in feedstuffs in Portugal: an overview

Mycotoxins are secondary metabolites produced by many genera of fungi in many commodities, under certain conditions. Mycotoxicological control of feed is a procedure that aims to protect human and animal health, avoiding the adverse effects of these undesirable substances. This component of the sanitary control of feed and food is essential to prevent the presence of those substances which can seriously affect the health of the animals. In Portugal, there is relatively few information related to the natural occurrence of mycotoxins in feed. In this context, the authors present results and data compilation concerning the occurrence of mycotoxins in raw materials and also feed for dairy cattle, swine, poultry, horses, fish, laboratory rats and pet; making a generic qualitative appreciation of the risks associated to the presence of mycotoxins in these feedstuffs. The mycotoxins studied: aflatoxins (AFs), ochratoxin A (OTA), fumonisin B₁ and B₂ (FB₁, FB₂) were analysed by High Performance Liquid Chromatoghraphy (HPLC). Deoxynivalenol (DON) and zearalenone (ZEN) were determined by Thin-Layer Chromatography (TLC). The results suggest that contaminations with these mycotoxins in feed are quite common, revealing the need for surveillance and monitoring programs for the prevention of the sanitary impacts of these “non desirable substances”.     

Mycoflora and mycotoxins natural occurrence in corn from entre rios province, Argentina

Corn samples were collected in 1999 from three departments of Entre Réos province, Argentina, and were surveyed for mould contamination and natural occurrence ofFusarium mycotoxins, ochratoxin A and aflatoxins.Fusarium verticillioides was the most prevalent fungal species recorded at all departments. Zearalenone, deoxynivalenol and ochratoxin A were not found in any samples. Only one of the 52 corn samples analysed was contaminated with aflatoxin B₁ (17 μg/kg). Fumonisin B₁ was found in 58 % of samples (range of positive samples: 47– 3,347 μg/kg), fumonisin B₂ in 33.0 % (range of positive samples: 23–537 μg/kg) and fumonisin B₃ in 25.0 % (range of positive samples: 24–287 μg/kg) of them. This is the first report on the natural occurrence of mycotoxins in corn from Entre Ríos province, Argentina. Levels of fumonisins were lower than detected in other Argentinian provinces.     

In situ absorption of aflatoxins in rat small intestine

To evaluate the rate at which the four main aflatoxins (aflatoxins B₁, B₂, G₁ and G₂) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (k _a ) of 5.84±0.05 (aflatoxin B₁), 4.06±0.09 (aflatoxin B₂), 2.09±0.03 (aflatoxin G₁) and 1.58±0.04 (aflatoxin G₂) h^–1, respectively.     

Seasonal variation in exposure frequency and concentration levels of aflatoxins and ochratoxins in urine samples of boys and girls

Urine samples from children in Sierra Leone (134 boys and 110 girls), were collected during the dry season. During the rainy season samples were collected from 97 boys and 93 girls. Analysis of the dry season samples, revealed that, with the exception of one boy, all children had detectable amounts of aflatoxins and/or ochratoxins in their urine. Similarly, with the exception of four children (two from each sex), rainy season urine samples also contained these two mycotoxins. There were significant differences in the frequency of exposure to some mycotoxins: ochratoxin A (OTA), p < 0.01;4-hydroxyochratoxin A (4R-OTA), p < 0.002; aflatoxin M1 (AFM₁), p < 0.04;aflatoxicol (AFL), p < 0.03; aflatoxin B₂ (AFB₂), p < 0.04 . There were also significant differences in the levels of aflatoxin B₁ (AFB₁), (p < 0.05) and AFB₂, (p < 0.02) detected in dry season samples. Stratification of these results according to season and sex, has indicated significant differences with respect to 4R-OTA (p < 0.04) and AFB₁ (p < 0.02). The results of this study show that in Sierra Leone, children are frequently and constantly exposed to both aflatoxins and ochratoxins. This revised version was published online in August 2006 with corrections to the Cover Date.     

Detection and estimation of aflatoxins using both chemical and biological techniques

Production of aflatoxins on both natural (rice and corn) and semisynthetic (YES) media was conducted using an identified toxin-producing strain ofAspergillus flavus. TheA flavus strain was able to produce 4 types of aflatoxins, namely B₁, B₂, G₁, and G₂ on rice, corn, and YES media. Quantitative data showed that the concentrations of aflatoxins B₁ and G₁ produced were 52, 40.3, and 39.6; and 64.7, 45.0, and 58.0jug for 50g of rice, corn, and YES media, respectively. In comparison, the yielded amounts of aflatoxins B₂ and G₂ were much lower: 11.5, 17.9, and 17.5; and 28.S, 40.3, and 39.5 μg for 50 g of rice, corn, and YES media, respectively. A bioassay was conducted using the following 5 standard bacterial strains:Bacillus megaterium. Bacillus subtilis, Streptococcus faecal is, Staphylococcus epidermidis, andParacoccus denitrificans as well as a field strain of Candida albicans. All strains exceptP denitrificans showed varied degrees of inhibition when applied with crude aflatoxins at 5 to 40μg/mL. The minimum concentration of crude aflatoxins needed to inhibitP denitrificans was 10μg/mL. Moreover,Candida albicans was not inhibited at any concentration of aflatoxins applied in this work. Both undiluted and diluted (1/10, 1/100, and 1/1000) bacterial broth cultures showed a direct relationship between the diameter of inhibition zones and the concentrations of crude aflatoxins. Mean diameters of (7.0–20.5), (5–14), (4.5–13.0), (3.0–12.0), and (1.5–11.0) mm were observed when various concentrations of aflatoxins were applied usingB megaterium, S epidermidis, S faecal is, B subtilis, andP denitrificans, respectively. Field trials were applied to testify the validity of our data. A 1/100 dilution was prepared from each strain of 4 different species to estimate aflatoxins in samples of contaminated corn. Both chemical and biological assays were carried out at the same time. Data revealed that the most sensitive organism inhibited by as low as 7.5μg aflatoxins/mL wasB megaterium giving an inhibition zone of 10.5 mm, followed byS epidermidis with an inhibition zone of 7.5mm. In relation, the other 2 organisms were less sensitive to crude aflatoxins. Similarly, the biological assay was applied to detect aflatoxins in some samples of wheat, corn, peanut, rice, and poultry rations. Of the 14 wheat and 10 corn samples, only 4 wheat and 2 corn samples were found to be positive. The same results were obtained using TLC analysis.     

Natural occurrence of mycotoxins in cereals

Besides peanuts and cottonseed, cereal grains are the most important feed and food source that occasionally are naturally contaminated with mycotoxins. The problem of mycotoxins occurring naturally in cereals, especially in corn, has become trouble-some because of changing agricultural technology. The mycotoxin problem in cereals is not restricted to any geographic or climatic region. Toxins are produced on cereals, both in the field and in storage; they involve both the grain and the whole plant. The genera of fungi most involved areAspergillus, Fusarium, Penicillium andClaviceps. Mycotoxins known to occur naturally in cereals include aflatoxins B₁, B₂, G₁ and G₂-as well as aflatoxins M₁ and M₂-ochratoxins A and B, penicillic acid, patulin, ergot, zearalenone, citrinin, T-2, tenuazonic acid, kojic acid and sterigmatocystin. Of these mycotoxins, aflatoxins, patulin, penicillic acid and sterigmatocystin are carcinogens.     

Detoxification of mycotoxins by probiotic preparation for broiler chickens

Biological decontamination of mycotoxins using microorganisms is one of the well known strategies for the management of mycotoxins in foods and feeds. Among the different potential decontaminating microorganisms,Saccharomyces cerevisiae and lactic acid bacteria represent unique groups, which are widely used in food fermentation and preservation. The aim of this study was to determine the influence of spontaneous fermentation with the use of probiotic bacteria and yeast (Lactobacillus paracasei/casei ŁOCK 0920,L. brevis ŁOCK 0944,L. plantarum ŁOCK 0945,Saccharomyces cerevisiae ŁOCK 0142), on reduction of sum of aflatoxines (B₁, B₂, G₁, G₂) and ochratoxin A concentration during fermentation and the microflora pattern during fermentaton. The probiotic bacteria and yeast applied creates a starter culture for flour fermentation that has a stable feature of detoxication of aflatoxines and especially ochratoxin A. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Einsatz einer kombinierten Immunoaffinitätssäule zum Nachweis von Aflatoxinen (B<Subscript>1</Subscript>, G<Subscript>1</Subscript>, B<Subscript>2</Subscript>, G<Subscript>2</Subscript>), Ochratoxin und Zearalenon

A method for the combined determination of the mycotoxins aflatoxin B₁, G₁, B₂, G₂, ochratoxin A and zearalenone in cereals and feed is described. After extraction with acetonitrile/water or methanol/water the cleaning takes place with new combined immunoaffinity clean-up column “AflaOchraZea” by VICAM. When the mycotoxins are determined in different cereals with this new type of clean-up column low detection limits and high recovery rates can be reached similar to those obtained by using separate immunoaffinity clean-up colums for the said mycotoxins.     

Production of extracellular enzymes and aflatoxins in solid substrate fermentation with aflatoxigenic<Emphasis Type="Italic">Aspergillus</Emphasis> spp.

AflatoxigenicAspergillus flavus andAspergillus parasiticus were subjected to solid substrate fermentation process for 6 days to determine the formation of aflatoxins and production of extracellular enzymes (amyloglucosidase, cellulase, invertase and proteinase). Both organisms produced enzymes which generally increased with fermentation.Aspergillus flavus produced four enzymes whereasA. parasiticus produced three with no proteinase activity.Aspergillus parasiticus produced aflatoxins B₁, B₂ and G₁ but no G₂ andA. flavus produced aflatoxins B₁ and B₂. Invertase showed the highest activity withA. parasiticus and that corresponded with the highest total toxin produced. The enzyme activities were higher withA. parasiticus thanA. flavus although total toxins produced byA. parasiticus were lower than total toxins produced byA. flavus under the same environmental conditions.     

Mycoflora and mycotoxins of peanut (Arachis hypogaea L.) seeds in Egypt. III. Cellulose-decomposing and mycotoxin-producing fungi

From 40 peanut seed samples collected in Egypt, forty-three species and one variety of fungi, belonging to 16 genera, were collected. The most dominant genera were Aspergillus (11 species + one variety), Penicillium (11 species) and Fusarium (4 species). From the preceding genera A. fumigatus, A. flavus, A. niger, P. chrysogenum and F. oxysporum were the most frequent species.Forty-nine isolates belonging to 12 species and one variety were tested for production of mycotoxins, after growth on liquid medium containing two carbon sources (sucrose or cellulose). Thin layer chromatographic analysis revealed that the quality and quantity of mycotoxins was higher on sucrose than cellulose. Mycotoxins identified were aflatoxins B₁, B₂, G₁ & G₂, citrinin; fumagillin; diacetoxyscirpenol T-2 toxin; satratoxin H; and zearalenone.     

Fungal flora and aflatoxin contamination of feedstuff samples in Beida Governorate,Libya

Fungi of 19 genera, 30 species, and one variety were isolated from 25 samples of sheep-, cattle- and camel feedstuffs collected from different farms in the Beida Governorate, Libya.Aspergillus, Penicillium andFusarium were the most common genera in the three substrates tested. TLC was used to establish the identity of aflatoxins in the chloroform extract of all samples and the ability to produce aflatoxins byAspergillus flavus in a synthetic liquid medium. Twenty samples out of 25 tested were naturally contaminated and 21 isolates ofA. flavus out of 30 produced at least one of the following aflatoxins: B₁; B₁, G₁; and B₁, B₂, G₁, G₂. This is the first report about the natural occurrence of aflatoxins and aflatoxin-producers of the genusAspergillus in Libya.     

Mycotoxins producing fungi and mycoflora of air-dust from Taif,Saudi Arabia

Using the dilution plate method, 70 species and 31 genera were collected from 20 dust samples on glucose (28 genera and 64 species) and cellulose Czapek's agar (22 genera and 46 species) at 28° C. The most common fungi were Aspergillus niger, A. flavus, A. flavus var. columnaris, Phoma glomerata, Fusarium oxysporum, Penicillium chrysogenum and Mucor racemosus; and A. nidulans, Phoma humicola, Drechslera spicifera and Stachybotrys chartarum on the two media, respectively.Toxicity test showed that about 85% of the isolates tested were toxic to brine shrimp (Artemia salina). Thin layer chromatographic analysis revealed that 13 out of 23 toxic isolates produced known mycotoxins. Toxins identified were: aflatoxins B₁ and B₂, Kojic acid and trichodermine.     

Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

Examination of fungal growth and aflatoxin production on marihuana

Under favorable growth conditions,Aspergillus flavus andA. parasiticus produced aflatoxins on marihuana. Cultures ofA. flavus ATCC 15548 produced both aflat oxin B₁(AFB₁) and G₁(AFG₁). The production of AFG₁ was substantially greater than that of AFB₁. Cultures ofA. flavus NRRL 3251 andA. parasiticus NRRL 2999 produced only AFB₁. All natural flora cultures tested negative for aflatoxins. NoAspergilli sporulations were observed in these cultures. In the cultures inoculated with known toxigenic fungi, the highest mean level for total aflatoxins was 8.7 g/g of medium. Marihuana appears not to yield large quantities of these mycotoxins but sufficient levels are present to be a potential health hazard for both the user and the forensic analyst who is in daily contact with such plant material. Careful processing, storage, and sanitation procedures should be maintained with marihuana. If these conditions are disregarded due to the illicit status of marihuana, the potential for mycotoxin contamination must be considered.     

Fungal populations and mycotoxins in silage in Assiut and Sohag governorates in Egypt,with a special reference to characteristic Aspergilli toxins

Forty silage samples were collected from Assiut and Sohag governorates in Egypt to measure the presence of fungal population in silage. Forty-three species and 2 species varieties belonging to 17 genera were isolated using glucose Czapeks and Sabourauds dextrose agar media at 28 ^°C. The most prevalent genera were Aspergillus (57.5 and 100 of the samples), Penicillium (100 and 55%) on the two mentioned media, respectively. Also, Fusarium oxysporum and Gibberella fujikurori were recovered in moderate incidences. Mycotoxin profiles were also determined in these samples: Aflatoxins showed the highest incidence rates of occurrence, it occurred in 22.5% of all samples analyzed. Other mycotoxins were detected from all samples (T₂ toxins and sterigmatocystin at incidence of 7.5 and 5%, respectively). The screening of the characteristics mycotoxins of different isolates of Aspergillus isolated from silage samples was tested. The results clarified that some mycotoxins (aflatoxins – aspergillic acid – beta nitro propionic acid – cyclopiazonic acid– kojic acid and sterigmatocystin) were produced by some isolates of A. flavus. Some isolates of A.fumigatus could produce gliotoxin and verrucologen. All of A. niger isolates tested were able to produce kojic acid. One isolate of A. ochraceous formed ochratoxin A and other isolate produced penicillic acid. Concerning A. terreus isolates, the results showed that 5 isolates were able to produce citrinin and 4 isolates had ability to produce patulin. A. versicolor isolates showed the ability to produce ochratoxin A.     

The mycotoxin research in foodstuffs in the Czech Republic in the 90th Years

Mycotoxins are naturally occurring secondary metabolites produced by several toxigenic microscopic fungi on a variety of crops, especially cereal grains and further foodstuffs. Series of experimental research projects on the determination of mycotoxins (aflatoxins, cyclopiazonic acid, ochratoxin A, patulin, deoxynivalenol, fumonisin B₁, T-2 toxin, zearalenone, sterigmatocystin, alternaria toxins) in several foods were realized in the National Reference Centre for Microscopic Fungi and Mycotoxins in the 90^th years. The aim of our work was an estimation of dietary exposure to mycotoxins and risk assessment. The method of a solid phase extraction (SPE), liquid — liquid extraction and immunoaffinity chromatography (f. e. R-Biopharm, VICAM) were used to elaborate for sample analyses of mycotoxins in our projects. The mycotoxins were detected most frequently by chromatographic methods (HPTLC, HPLC, GC) and immunochemical methods (ELISA). Average dietary exposure has been calculated by multiplying of concentration data for specific foods with their consumption rates per 1 kg of b. w. per day. The estimation of the dietary exposure dose of mycotoxins for the Czech population is presented.