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Influence of harvesting and drying techniques on microflora and mycotoxin contamination of figs.

Mould growth and mycotoxin (aflatoxins and ochratoxin A) formation were examined in the 1993 dried figs crop. The relationships between mould/mycotoxin contamination and orchard conditions, different harvesting techniques, harvesting time and intactness of fruits were investigated. The fruits were examined during drying and effects of different pretreatments, sun drying and solar drying on the mould and mycotoxin contamination in figs were also studied. Aflatoxins (B1, B2, G1 and G2) were not present in the firm or shrivelled ripe figs. Among the samples examined during drying, only one of the 32 samples was found to be aflatoxin positive. Ochratoxin A was not detected in any of the samples analysed. The moisture content, aw and pH values of full ripe and shrivelled fruits were suitable for mould growth and mycotoxin formation while these parameters in pretreated and dried fruits were found to be too low to allow such outcome. It was observed that harvesting the fruit by hand-treating with different solutions and application of solar drying were effective in reducing contamination level.

Aflatoxins↗

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 micrograms/kg and one sample each of flour and bread at concentrations 188 and 179 micrograms/kg. Zearalenone (ZEN) was detected in ten samples of wheat at levels from 28 to 42 micrograms/kg and four samples each of flour and bread at concentrations of 95 and 34 micrograms/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 micrograms/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 micrograms/kg for wheat and to 125, 45 and 1.0 micrograms/kg for flour after 4 kGy exposure and a sharp drop in Fusarium toxin levels occurred at 6 kGy and as eliminated at 8 kGy. Bread prepared from 6 kGy was contaminated with Fusarium toxin at levels below 5 microgram/kg. It was noticed that gamma-irradiation reduce greatly the natural occurrence of Fusarium mycotoxins in bread.

Bread↗

Advantages and drawbacks of immunoaffinity columns in analysis of mycotoxins in food.

A number of countries are setting legislations on mycotoxins. In order to reduce dispute between importing and exporting countries, the analytical data should be as comparable as possible, especially when levels are close to the regulatory limits. The present trend in the analysis of mycotoxins is to use immunoaffinity column (IAC) as a clean-up and enrichment technique, and Association of Official Analytical Chemists and European Union have validated methods which address a few food commodities. This study describes our experience using both conventional and IAC approaches in the analysis of three mycotoxins. Aflatoxins (AFs): Aflatoxin G1 has been detected by liquid-liquid partitioning methods with HPLC detection as false-positive in some maize. On IACs, this compound behaves as an AF, lowering the amount of the AFs trapped. The problem was solved using either TLC or HPLC with detection in the Kobra cell. Depending on the additives to food during the processing and cooking, the AFs might appear as an opened ring not recognised by the antibody. Fumonisins (FB): Compounds interfering with the FB's antibodies were also observed while analysing breakfast cereals leading to underestimation of FB. Ochratoxin A (OTA): Depending on the food composition and extraction techniques, OTA is underestimated with IAC in some breakfast cereals and coffee. These data strengthen the necessity to validate methods using IAC for each complex matrix.

Aflatoxin B1↗

Design of sampling plans for mycotoxins in foods and feeds.

The control of the occurrence of mycotoxins in foods and feeds requires effective surveillance and quality control procedures which facilitate the identification and control of the mycotoxin problem respectively. Surveillance and quality control procedures involve a sequence of sampling, sample preparation, and analysis steps; and the integrity of the data produced by these procedures will be determined by the effectiveness of these steps. It is imperative that the sampling step is performed as accurately as possible so that the sample collected is representative of the batch of food or feed under investigation. Needless to say, the collection of a biased sample will completely invalidate the resultant analytical data. Most attempts to develop effective sampling protocols have focused upon the aflatoxins, since the majority of current regulations are concerned specifically with this group of mycotoxins. However, the design of effective sampling protocols has been severely hindered by the highly skewed distribution of the aflatoxins in foods and feeds. Studies already performed indicate that representative samples of commodities, composed of large particles (e.g., corn and oilseed kernels) should be 10 kg in weight, at least, and composed of approximately one hundred incremental samples. Similar studies have indicated that samples of oilseed cakes and meal, however, should be composed of fifty incremental samples which afford a composite sample of approximately 5 kg in weight.

Food Analysis↗

Keeping the mycotoxins out: experience gathered by an international food company.

As early as the mid-1960s the analysis of aflatoxins became an important part of Nestlé's quality control programme, especially since the Company's products include a wide variety of milk- and cereal-based foods intended for infants and children. At that time, dealing with this problem was hampered by the lack of simple and reliable analytical methods, the absence of legal provisions in many countries, the widely different origin of the raw materials, and the fact that sources of contamination were often beyond the Company's control. However, over the years substantial progress has been made, mainly by setting up an inspection system including suitable methods of analysis that allow compliance with raw material specifications to be checked at source. In spite of the fact that the problem of aflatoxins in milk, cereals, and oilseeds has now been largely tackled, it is increasingly necessary to step up our mycotoxin surveillance programme. Indeed, several other mycotoxins have now become a matter of concern, as witnessed by the recent fumonisin scare. Moreover, increasingly severe legislations put a heavy burden on the analysts responsible for the inspection of the raw materials, especially in European countries. This article presents the Company's considerable efforts over a period of almost 30 years in trying to keep the mycotoxins out, or at least to limit their concentrations to the lowest possible levels.

Food Analysis↗

Some reflections on establishing a community legislation on mycotoxins.

A Community legislation on mycotoxins is essential to protect public health and to avoid trade barriers and competition distortions. Maximum permitted levels in food are to be fixed. Some orientations for this purpose are indicated. The verification of the compliance with maximum permitted levels shall need suitable analytical methods. Up to now, such methods were fixed by directives. This approach, however, had some disadvantages. A more flexible approach is to be planned. Among the mycotoxins to be taken into consideration, priority should be granted to the aflatoxins and to ochratoxin A. Fumonisins also deserve interest. Other mycotoxins will be taken into consideration as the need arises and scientific data are available. Some basic remarks on the rationale for establishing maximum permitted levels in food are made.

Food Analysis↗

New possibilities of matrix-assisted laser desorption ionization time of flight mass spectrometry to analyze barley malt quality. Highly sensitive detection of mycotoxins.

The occurrence of mycotoxins in agricultural commodities is a major health concern for livestock, humans, and the environment. Barley and subsequently malt quality is of fundamental importance to obtain good quality beer. Classical methods of analysis often require tedious, laborious, and expensive processes. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) instrumentation enables highly sensitive and fast analysis and/or detection using a very small sample. The possibilities of MALDI-TOF MS for he identification and/or detection of trichothecene mycotoxins (deoxynivalenol (DON) and nivalenol (NIV), respectively) in barley malt were examined, and it was found that almost all classical MALDI matrices failed to ionize the compounds being studied. This detailed study of the ionization conditions and the search for unconventional matrices led to the discovery of suitable MALDI matrices, which enable ionization of trichothecene mycotoxins. These were: fine powdered synthetic diamond, sodium azide, or hydrazine hydrate. It is possible to detect 8.5 x 10(-12) mol (2.5 ng) of deoxynivalenol or 64 x 10(-12) mol (20 ng) of nivalenol in just 1 microL of barley malt extract (equivalent to 600 microg of DON in 1 kg of barley malt). The procedure developed enables fast determination of DON and NIV in barley, malt, or similar products.

Beer↗

Mycotoxin-induced elevation of free sphingoid bases in precision-cut rat liver slices: specificity of the response and structure-activity relationships.

Fumonisin B1 (FB1) is the predominant member of a family of toxic metabolites produced by several species of Fusarium and is commonly found on corn. FB1 is a potent competitive inhibitor of ceramide synthase, which catalyzes the conversion of sphinganine and sphingosine to ceramide. The resultant accumulation of free sphingoid bases and the disruption of sphingolipid metabolism is believed to be the mechanism of toxicity of the fumonisins. The objectives of this study were to determine the relative potency of analogs of FB1 to inhibit ceramide synthase and to determine whether the inhibition is specific to mycotoxins with fumonisin-like structures. Fumonisins B1, B2, B3, B4, C4, and TA toxin (a structurally similar mycotoxin produced by the tomato pathogen, Alternaria alternata f. sp. lycopersici) were approximately equipotent inhibitors. Hydrolyzed fumonisins B1, B2, and B3, which lack the tricarballylic side chains, were only 30-40% as potent as the parent toxins. N-acetylated FB1 (FA1) did not block ceramide synthase, suggesting that FA1 is nontoxic. Inhibition of ceramide synthase by fumonisin analogs did not appear to be related to the lipophilicity of the compounds, as determined by computer estimation of log P values. The ability of relatively high (10 and 100 microm) doses of other mycotoxins that bear no structural similarity to fumonisins, including aflatoxin B1, cyclopiazonic acid, beauvericin, T-2 toxin, sterigmatocystin, luteoskyrin, verrucarin A, scirpentriol, and zearalenone, to block ceramide synthase was also determined. All of the toxins tested were negative in the bioassay with the exception of fumonisins, indicating that disruption of sphingolipid metabolism is a specific cytotoxic response.

Acetylation↗

Mycotoxins as carcinogens.

Fungi which contaminate food and feed products produce toxic metabolites under favourable conditions, thus posing a serious health problem. A variety of mycotoxins are produced by different fungi, but aflatoxins have been investigated more extensively. Varied reports are available on the production of aflatoxins by different species of Aspergillus and Penicillium; however, A. flavus and A. parasiticus have the greatest capacity and appear to be the primary sources of aflatoxins. Preformed mycotoxins are ingested by man through the consumption of vegetable and animal products (milk, meat, etc.). Factors affecting toxicity include species, age, sex, and nutrition. Evidence from animal experiments provides enough information to indicate that most of the mycotoxins are carcinogens.

Aflatoxins↗

Use of cell cultures for predicting the biological effects of mycotoxins.

The risk presented by mycotoxins is a toxicological problem. As the data given by physico-chemical analysis may be difficult to translate in terms of toxicity, however, especially when considering multiple contamination, we have developed a system for toxicological analysis of mycotoxins using cell cultures of different origins. The response of several cell types to a number of well defined mycotoxins was obtained in three days. This approach allowed us to: demonstrate and quantify a toxic effect, define some organ specificity related to the preferential action on a particular cell type, and detect an immunosuppressive effect. The results indicate that the system can be used for toxicological screening and that it has a predictive value for the pathological effects of tested products.

Animals↗

Trichothecene mycotoxins in the dust of ventilation systems in office buildings.

Analysis of trichothecene mycotoxins in dust samples from ventilation systems of office buildings was applied as a rapid and inexpensive method for the detection of mycotoxins. Dust samples from three different office spaces of the Montreal urban area, reportedly affected by the "sick buildings syndrome", were analysed by thin-layer chromatography (TLC). Positive colour reaction on TLC plates with 4-(p-nitrobenzyl) pyridine, specific for the 12,13-epoxy group in the trichothecene nucleus, was obtained for the extracts of 0.5- to 50-g dust samples. The dust samples contained at least four trichothecenes: T-2 toxin, diacetoxyscirpenol, roridine A and T-2 tetraol. The results were confirmed by high-performance liquid chromatography analysis. Screening of dust samples from air ventilation systems of reportedly affected buildings provided direct evidence of trichothecene mycotoxins, with the detection limit estimated as 0.4-4 ng/mg dust. Thus, the dust sample analysis is suggested as a rapid technique for detecting the presence of myotoxins in the dust of ventilation systems.

Air Pollution, Indoor↗

Chromatography of mycotoxins on precoated reverse-phase thin-layer plates.

Reverse-phase thin-layer chromatography of 18 common mycotoxins was carried out on Merck C18 and Whatman C18 and diphenyl bonded phases. The mobile phases included neutral and acidified binary mixtures of water and methanol, acetonitrile or tetrahydrofuran. Mycotoxins studied included aflatoxins B1, B2, G1, G2, sterigmatocystin, ochratoxin A, citrinin, penicillic acid, patulin, zearalenone, and the trichothecenes diacetoxyscirpenol, HT-2 toxin, nivalenol, neosolaniol, fusarenone-X, T-2 toxin, deoxynivalenol and 3-acetyl-deoxynivalenol. Both C18-bonded phases performed well, and could serve as a convenient confirmation for mycotoxins appearing in normal-phase (silica) thin-layer chromatographic screening procedures.

Chromatography, Thin Layer↗

[A screening method for the determination of various mycotoxins in food (author's transl)].

A simple analytical method for multiple mycotoxins was developed for detecting aflatoxin B1, B2, G1, G2, M1, and M2, sterigmatocystin, penicillin acid, ochratoxin A and patulin. These mycotoxins were extracted with ethyl acetate. The extract was cleaned up by chromatography on silica mini-column. Each fraction was separated by thin-layer chromatography. The final evaluation of the TLC-plates was carried out by fluorodensitometry. A considerable increase in fluorescence intensity was achieved by spraying the plates with paraffin after development. The detection limits of fluorescent mycotoxins and the fluorescent derivates of penicillic acid and sterigmatocystin were lowered to 1:10 up to 1:100. Spots of patulin were measured by fluorescence quenching.

Aflatoxins↗

Production of tremorgenic mycotoxins by isolates of Aspergillus fumigatus from sawmills in Sweden.

One hundred and six strains of A. fumigatus were isolated from 21 sawmills in Sweden, and 73 of these strains were examined for production of fumitremorgen B and verruculogen (tremorgenic mycotoxins) on YES-medium using thin layer chromatography (TLC). Twenty-three strains (32%) were tremorgen producers and 50 strains (68%) were non-producers. Tremorgenic mycotoxins were detected in conidia of seven A. fumigatus strains. The amount of toxin varied between 0.6-8.0 microgram/10(8) conidia (mean value 2.3 micrograms/10(8) conidia, equivalent with 0.18%). No production of the mycotoxin gliotoxin was detected in 6 strains of A. fumigatus. No tremorgens were detected during mould growth on wood substrates, in spite of the use of different wood species (Scots pine, Pinus sylvestris: Norway spruce, Picea abies and birch, Betula spp.), dried versus non-dried wood, bark (pine), leached wood, and wood after various sterilization methods.

Air Pollutants↗

Mycotoxin producing potential of Fusarium graminearum isolates from Uruguayan barley.

Twelve isolates of Fusarium graminearum were obtained from barely grains collected from different Uruguayan regions (harvest 1993-94). This was the predominant fungal species contaminating the crop due to a particular humid and warm season with cold nights conductive to toxin production The isolates were grown on moist, sterile rice, extracted with aqueous methanol, and examined for mycotoxin production. Zearalenone (ZEA) and the trichothecenes deoxynivalenol (DON), 3- and 15-acetyl-DON (AcDON), nivalenol (NIV), nivalenol (NIV), fusarenon-X (FX) and T-2 toxin (T-2) were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) and confirmed by gas chromatography-mass spectrometry (GC-MS). Eleven of the 12 strains were DON and/or ZEA producers and 9 were AcDON positive. No NIV, or FX were detected. One strain produced T-2. The predominant acetyl-DON isomer was 15-AcDON. Mass-spectral analysis yielded detectable levels of other mycotoxins, 13-OH-apotrichothecenes, 11-epiapotrichothecenes, culmorin, sambucinol, and isotrichodermol being the most numerous. From the metabolic profiles it is suggested that Uruguayan F. graminearum strains belong to the chemotype IB (DON/15-AcDON). The predominance of this chemotype is in accordance with data from Canada, United States, Mexico and Argentina which have similar climatic conditions that would favor F. graminearum growth and mycotoxin production.

Fusarium↗

Ear-rot fungi and mycotoxins in South African corn of the 1989 crop exported to Taiwan.

A shipment of South African corn (1989) exported to Taiwan, was analyzed for various ear-rot fungi and Fusarium mycotoxins. Two sets of samples, one from the points of origin in South Africa prior to shipment, and the other from the end-point distributors in Taiwan, were studied. Surface-sterilized kernels were plated onto two different agar media and the fungal colonies identified. High Performance Liquid Chromatography was used to analyze mycotoxin levels. The predominant ear-rot fungi, in decreasing order of isolation frequency, were Fusarium subglutinans, F. moniliforme, Diploidia maydis and F. graminearum. Aspergillus flavus and A. parasiticus were not isolated from samples prior to export, but a small number of A. flavus isolates were found after shipment. The predominant mycotoxins were fumonisins B1 (0-865 ng/g) and B2 (0-250 ng/g). Low levels of moniliformin (< or = 390 ng/g) were detected in some samples before shipment. Zearalenone (25 ng/g), and nivalenol (120 ng/g) were detected in two out of 32 samples taken in Taiwan. The samples contained no detectable levels of either aflatoxins (> 0.5 ng/g) or deoxynivalenol (> 100 ng/g) before or after shipment.

Aspergillus↗

[Effect of the fungicide matador (tebuconazole/triadimenol) on mycotoxin production by Fusarium culmorum].

Applications of the fungicide Matador to winter wheat (Slejpner) inoculated at earing with Fusarium culmorum resulted in a considerable decrease in the incidence of Fusarium headblight. In the study presented here, subsequent mycotoxin analyses by selected ion monitoring gas chromatography-mass spectrometry revealed higher levels of nivalenol (NIV) in samples singly treated with Matador at 1 L/ha. Amounts as high as 2432 micrograms NIV/kg and 860 micrograms NIV/kg, representing a 16- or six-fold increase as compared to controls, were found in wheat treated with the fungicide 3 h before or 24 h after inoculation of the grain with F. culmorum, respectively. In parallel, higher cytotoxic activities of these samples were obtained when testing crude sample extracts in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-cell culture bioassay using swine kidney monolayers as target cells. It is concluded that treatment of grain with the fungicide Matador could result in a marked stimulation of the production of NIV by F. culmorum. As the incidence of Fusarium headblight did not correlate with the amount of mycotoxins found, the need for mycotoxin analyses combined with bioassays such as the MTT-cell culture assay is recommended for a meaningful assessment of the quality of grain treated in such a manner.

Animals↗

[A cell-culture assay for the detection of mycotoxins. I. Investigations with pure toxins (author's transl)].

For the detection of mycotoxins by a biological assay cells were seeded in Microtiter-plates together with toxin. After 24 h and 6 days of culture cells were examined visually for cytocidal effects, growth inhibition, and morphological aberrations. Three human and 2 porcine cell-lines were compared as to their reaction with 16 mycotoxins and 9 organic chemicals. The results indicate that the toxin-activating systems differ with type and origin of cells. This suggests the use of several selected cell-lines in parallel for testing of microbially derived food additives for possible contamination by mycotoxins.

Animals↗