Mycotoxin formation in moist 2-row and 6-row barley during granary storage

Eleven-kilogram parcels of 2-row and 6-row barley initially at 18% moisture content were implanted in dry bulk oats in a farm granary in Manitoba for 60 weeks between August 1983 and October 1984. Temperature, moisture content, O₂ and CO₂ levels, fat acidity values, seed germination, microfloral incidence and abundance and the presence of major mycotoxins (aflatoxins, sterigmatocystin, ochratoxin A, citrinin, penicillinic acid, patulin) were monitored. Ochratoxin A reached maximum levels of 0.97 ppm by week 24 in the 6-row barley, and 0.05 ppm by week 28 in the 2-row; no other mycotoxins were detected. The effect of cultivar type was significant (P<0.01) with greater effects in the 6-row barley for the following parameters: fat acidity value, germination, incidence of Alternaria alternata, Penicillium spp. and Helminthosporium sativum, total fungal propagule count and ochratoxin A levels. The effect of time was significant (P<0.05) for all variables except oxygen, carbon dioxide, Aspergillus versicolor, and total fungal propagule count. The interaction between cultivar and time was significant (P<0.01) for Alternaria and Helminthosporium only.Contribution No. 1233, Agriculture Canada Research Station, Winnipeg, Manitoba R3T 2M9, Canada.     

Microflora and condition of flood-damaged grains in Manitoba,Canada

Microfloral and other changes which occurred in farm-stored grains at, below and above the maximum water level were documented after the Red River Valley flood of spring 1979 in southern Manitoba, Canada. Results of flood-damage simulation studies carried out in the laboratory on wheat, rapeseed and flax generally confirmed those from the field. In laboratory simulation studies with flooded grains in tanks a 2.5–10.0 cm thick crust of sprouted seeds developed just above the maximum water level. Migration of moisture occurred upwards from the base of the crust and maximum temperatures just beneath the crust. Beneath the crust was a horizon of putrid seeds of zero germination, and above the crust a horizon of visible post-harvest molds, e.g.Aspergillus flavus andPenicillium spp. and harvest molds, e.g.Trichothecium roseum. Penicillium verrucosum var.cyclopium occurred throughout the profile, whereasAlternaria alternata, Wallemia sebi andAspergillus glaucus group species were confined to upper horizons because of low seed moisture andA. flavus and A. versicolor to lower horizons of high moisture. Aflatoxins B₁ at 426 ppb and B₂ at 34 ppb were the only mycotoxins detected in either study, and occurred in a partially filled granary of flax which had been affected by flood waters. Pressure effects produced by the expanding seeds such as increase in height of grain mass and splitting of seams of granaries were evident in both studies. Rounded or slippery seeds, e.g. peas, flax, were associated usually with vertical pressure effects whereas irregularly-shaped seeds, e.g. wheat with lateral pressure effects. It is hoped that this work will provide a better understanding of the processes involved when grain is damaged by flood waters and assist in the development of improved salvage procedures.Contribution No. 942, Agriculture Canada Research Station, 195 Dafoe Road, Winnipeg, Manitoba, R3T 2M9.     

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.     

Quality changes in granary-stored wheat at 15 and 19% moisture content

Half-bushel parcels of wheat at 15 and 19% moisture content (MC) were implanted in bulks of dry stored oats in a farm granary for 60 weeks to monitor quality changes. Temperature, MC, fat acidity value (FAV), oxygen and carbon dioxide levels, germination, microfloral incidence and abundance, and major storage mycotoxins (aflatoxins, sterigmatocystin, ochratoxin A, citrinin, penicillic acid) were monitored at two-week or four-week intervals during storage. Wheat storage at 19% MC was characterized by higher temperature, MC, FAV, carbon dioxide level, and microfloral abundance, and lower levels of oxygen and germination as compared to 15% MC wheat; no mycotoxins were detected at either moisture. Approximately 19% MC may represent a critical moisture limit for mycotoxin formation in granary stored wheat, given the indigenous mycoflora.     

Mycotoxin formation in moist wheat under controlled temperatures

One-kilogram parcels of wheat with 20.5% moisture content were maintained at 15° and 22 °C for 10 weeks to study quality changes. Temperature, moisture, oxygen and carbon dioxide levels, microfloral incidence and abundance, seed germination, fat acidity values, aflatoxins, sterigmatocystin, ochratoxin A, penicillic acid, citrinin and zearalenone were monitored. By two weeks, trace levels of ochratoxin had formed at both temperatures. By 10 weeks, the wheat contained at least three times more ochratoxin A at 22 °C than at 15 °C. Strains of Penicillium verrucosum var. cyclopium were associated with ochratoxin A production. No other mycotoxins were detected. The effect of temperature was significant for all variables (greater effect at 22 °C) except A. glaucus gr. and Penicillium (P<.01). The effect of time was significant for all variables except bacteria (P<.01). The shape of the response was fully characterized by the linear and quadratic terms, except for % moisture which was linear only, and for bacteria for which time was not significant. The interaction between time and temperature was significant (P<.01) for total fungal propagule count, % moisture, and Aspergillus versicolor, indicative of the steeper rise in slope for 22 °C.     

Isolation and purification of an enzyme hydrolyzing ochratoxin A from <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Ochratoxin A is a mycotoxin produced by several Aspergillus and some Penicillium species which may be present in food and feed products. It can be enzymatically hydrolyzed into ochratoxin α and l-β-phenylalanine, thereby decreasing its toxicity. The ochratoxin A degradation capacity of Aspergillus niger is well known and here we report the isolation and purification of a novel enzyme from A. niger that hydrolyzes this mycotoxin. A wheat germ medium supplemented with ochratoxin A was used to produce the enzyme, which was purified from culture filtrate by acetone precipitation and anion exchange chromatography. An overall purification of 2.5-fold with a recovery of 68% and a final specific activity of 36 U/mg was obtained. The enzyme is a metalloenzyme as it was inhibited at 10 mM EDTA, whereas PMSF had no effect. The ochratoxin A hydrolytic enzyme presented a V _max of 0.44 μM/min and a K _m of 0.5 mM when the reaction was carried out at pH 7.5 and 37°C.     

Mycotoxigenic infestation in samples of cereal straw from Bihar, India

A survey of different types of cereal straw samples viz. paddy, maize and wheat, from Bihar State, India, was conducted in order to examine the mould flora and mycotoxin contamination. Out of 170 samples examined for mould flora,Aspergillus flavus group of fungi had highest level of incidence followed byA niger. Isolates ofA flavus, A ochraceus, Fusarium verticillioides andPenicillium citrinum were screened for their mycotoxins producing abilities. Out of 75, 63 and 68 isolates ofA flavus group obtained from stored straw of paddy, maize and wheat samples, respectively, 27 (36%), 14 (22%) and 24 (35%) were found to be toxigenic which produced different combinations of aflatoxins in different concentrations. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi from all types of samples. Out of 222 samples of straw analysed for natural occurrence of different mycotoxins, besides the aflatoxins present, zearalenone, ochratoxin A and citrinin were also recorded alone or as co-contaminants. A conducive climate together with the socioeconomic conditions of this region are important determinants for the high incidence of mycotoxins in cereal straw samples.     

Fusarium spp. and storage fungi in suboptimally stored wheat: Mycotoxins and influence on wheat gluten proteins

When wheat is stored under suboptimal conditions, a further mycotoxin increase of deoxynivalenol (DON), but especially of mycotoxins produced by storage fungi, e.g. ochratoxin A, is possible, lowering wheat quality and food safety. Different storage trials were conducted under suboptimal storage conditions.Fusarium survival during suboptimal storage was monitored by cultural technique and multiplex-PCR and set into relation to DON contents. Furthermore, XANES spectroscopy was applied on a selected storage trial in order to characterize sulfur speciation in low molecular weight (LMW) subunits of glutenin isolated from suboptimally stored wheat samples highly infected withFusarium and from wheat infected withAspergillus andPenicillium. Distinct changes in sulfur speciation were observed in grains infected with storage fungi, especially a significant increase of higher oxidation states (sulfoxide state, sulfonate state). Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Untersuchungen Zur Kühllagerung Von Weizen

Abstract

Saatweizen wurde ohne vorhergchende Sterilisation mit einem Ochratoxin A und Citrinin bildenden Stamm von Penicillium verrucosum beimpft und bei Wassergehalten von 18, 20, 22, 24 und 26% bei 4 und 10°C gelagert. Die Produktion von Ergosterin, eines chemischen Indikators für die von Pilzen gebildete Biomasse, setzte innerhalb der untersuchten Lagerdauer (240 Tage) ein. Lediglich bei 18% H₂O/4°C war keine Zunahme des Ergosteringehaltes zu beobachten. Ochratoxin A und Citrinin waren bei 18% H₂O/4°C und 20% H₂O/4°C über 240 Tage nicht zu finden (Nachweisgrenze: 10 bzw. 25 μg/kg). Bei den übrigen Kombinationen von Wassergehalt und Temperatur begann die Anhäufung der beiden Toxine etwa gleichzeitig mit der Ergosterinproduktion. Mit steigendem Wassergehalt und steigender Temperatur nahm die Zeit bis zum Beginn der Ergosterinproduktion ab, während die Geschwindigkeit der Produktion von Ergosterin, Ochratoxin A und Citrinin zunahm. Beide Toxine wurden während einer ersten Phase der Anhäufung etwa mit gleicher Rate gebildet. Bei 20–26% H₂O hatten der Wassergehalt und die Temperatur keinen Einfluß auf die Beziehung zwischen dem Toxingehalt und dem gleichzeitig erreichten Ergosteringehalt. Es wird empfohlen, stark mit Penicillium verrucosum kontaminierten Weizen nicht über den Beginn der Ergosterinproduktion hinaus zu lagem.

INVESTIGATIONS ON REFRIGERATED STORAGE OF WHEAT

1. Ergosterol, ochratoxin A and citrinin after inoculation with Penicillium verrucosum

Seed wheat was innoculated without having been sterilized with an ochratoxin A and citrinin forming strain of Penicillium verrucosum and stored at moisture contents of 18, 20, 22, 24, and 26% at 10 and 4°C. The production of ergosterol, a chemical indicator of fungal biomass, started within the storage time investigated (240 days). Only at 18% H₂O/4°C an increase of the ergosterol content was not observed. Ochratoxin A and Citrinin were not detected at 18% H₂O/4°C and 20% H₂O/4°C within 240 days (detection limit: 10 and 25 μg/kg, respectively). At the other combinations of moisture content and temperature the first detection of the two toxins approximately coincided with the onset of ergosterol production. With increasing moisture content and temperature the time up to the start of ergosterol production decreased, whereas the production rates of ergosterol, ochratoxin A and citrinin increased. Both toxins were produced with about the same rate during a first phase of accumulation. At 20–26% H₂O there was no influence of moisture content and temperature on the relation between toxin content and the simultaneously reached ergosterol content. It is recommended that wheat highly contaminated with Penicillium verrucosum should not be stored beyond the start of ergosterol production.     

Biological,physical and chemical changes in stored wheat

Six lots of wheat from six farmers' bins in Manitoba were adjusted to a range of 5 moisture contents, held at 6 temperatures and sampled at 6 times during storage. After sampling, seeds were surface sterilized with mercuric chloride and subsamples plated on Czapek's agar, on filter paper moistened with water, or on filter paper moistened with a 7.5% sodium chloride solution. The microflora on the seed was determined, germination counts were made, and the seed was examined by government inspectors for its condition and grade. A total of 1192 samples were examined. In addition, 180 samples were subjected to a fat acidity test. Deterioration for each combination of temperature and moisture, the fungi involved, and the consequent effect on condition, grade and fatty acid content were observed. The effect of high moisture content (>20%) and low temperatures (3–10 ° C) on infection of seed by Penicillium and the consequent effect on germination, condition, grade and fatty acid value are stressed. The interrelationship among fungi, and among fungi and temperature, moisture, storage time, fat acidity values and germination are indicated by correlation coefficient matrices.Deceased July 25, 1982.     

The use of yeast for microbial degradation of some selected mycotoxins

Several biodegradation experiments were carried out using 10 different yeast strains.Saccharomyces spp., Kluyveromyces spp. andRhodotorula spp. were tested for biodegradation of selected mycotoxins (ochratoxin A, nivalenol, deoxynivalenol and fumonisin B₁) standardsin vitro. There was confirmed that some yeast strains are able to degrade some mycotoxins. However, great differences between individual strains were observed. Moreover, 12Saccharomyces cerevisiae strains were tested for their potential capability to degrade zearalenone and fumonisins in Sabouraud broth. Two strains were capable to degrade zearalenone totally, one strain decreased the mycotoxin concentration up to 25%, and one strain up to 75% of original amount. Two strains were capable to degrade fumonisins partially.     

Mycotoxin production by Aspergillus niger aggregate strains isolated from harvested maize in three Portuguese regions

BackgroundMaize is considered one of the crops more susceptible to mycotoxins in the world. Two of the mycotoxins commonly associated with maize are fumonisins and ochratoxin A. Aspergillus niger is a known producer of ochratoxin A and is easily found in maize. Recently, however, A. niger has been reported to produce as well fumonisins, mainly fumonisin B₂.AimsThe aim of this study was to isolate A. niger strains from maize samples collected in three Portuguese maize growing regions and to detect the production of both fumonisin B₂ and ochratoxin A.MethodsNinety five maize samples were collected, plated, and all observable Aspergillus section Nigri strains were isolated. Strains were morphologically characterized and mycotoxin production was determined by HPLC-FD.ResultsIsolations resulted in a total of 270 strains of black Aspergillus from 73 samples (77% of the samples). About 14% of those strains were found to produce ochratoxin A and 39% of the strains were found to produce fumonisin B₂.ConclusionsAn association between the production of these two mycotoxins could not be found and no conclusions could be taken whether the presence of A. niger aggregate strains will increase the risk of maize contamination with fumonisins and more specifically with fumonisin B₂.     

Degradation of ochratoxin a and b by the white rot fungus<Emphasis Type="Italic">pleurotus ostreatus</Emphasis>

Degradation of ochratoxin A (OTA) and B (OTB) by three selected fungi during solid state fermentation of barley contaminated with ochratoxins was compared. In presence of the soil fungusRhizopus japonicus and the white rot fungusPanerochaete chrysosporium more than 60 % of the mycotoxins remained stable, while in the white rot fungusPleurotus ostreatus only 23 % of the initial OTA and 3 % of OTB were detected after a four weeks incubation period. Kinetic studies on mycotoxin degradation byPI ostreatus demonstrated formation of ochratoxin α and presumably ochratoxin β as intermediate products, what indicates that hydrolysis is the first step in OTA and OTB degradation followed by further degradation of the intermediates.     

Ochratoxin A induced TNFα release from the isolated and blood- free perfused rat liver

Ochratoxin A (OTA) a naturally occuring mycotoxin is nephrotoxic, hepatotoxic, teratogenic, embryotoxic, genotoxic, cancerogenic and immunosuppressive. Focussing on the immunemodulating effects, we found that OTA induces cytokine release from the isolated and bloodfree perfused rat liver. In the present study, we have characterized the liberation of tumor necrosis factor alpha (TNFα) by OTA and we described interactions with various mycotoxins andE.coli lipopolysaccharide (LPS).     

Natural Occurrence of Mycotoxins in Staple Cereals from Ethiopia

The occurrence of mycotoxins in barley, sorghum, teff (Eragrostis tef) and wheat from Ethiopia has been studied. Samples were analyzed for aflatoxin B₁ (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) using high performance liquid chromatography (HPLC) and for fumonisins (FUM) using enzyme linked immunosorbent assay (ELISA). AFB1 and OTA were detected in samples of all the four crops. AFB1 was detected in 8.8% of the 352 samples analyzed at concentrations ranging from trace to 26 μg kg⁻¹. OTA occurred in 24.3% of 321 samples at a mean concentration of 54.1 μg kg⁻¹ and a maximum of 2106 μg kg⁻¹. DON occurred in barley, sorghum and wheat at 40–2340 μg kg⁻¹ with an overall incidence of 48.8% among the 84 mainly ‘suspect’ samples analyzed; NIV was co-analyzed with DON and was detected at 40 μg kg⁻¹ in a wheat sample and at 50, 380, and 490 μg kg⁻¹ in three sorghum samples. FUM and ZEN occurred only in sorghum samples with low frequencies at concentrations reaching 2117 and 32 μg kg⁻¹, respectively. The analytical results indicate higher mycotoxin contamination in sorghum, which could be related to the widespread storage of sorghum grain in underground pits leading to elevated seed moisture contents. This is the first report on the occurrence of OTA in teff.     

Enniatin B and ochratoxin A in the blood serum of workers from the waste management setting

The waste management occupational environment is recognized by the simultaneous presence of several substances and biologic agents. Therefore, workers are exposed simultaneously to multiple contaminants. Occupational exposure to aflatoxin B₁ in one Portuguese waste sorting plant was already reported. However, besides this mycotoxin, data regarding fungal contamination showed that exposure to other mycotoxins could be expected. A study was developed to analyze if exposure to other mycotoxins besides aflatoxin B₁ was occurring in the workers from the waste sorting plant previously assessed and to discuss how these findings need to be considered in the risk assessment process. In addition to aflatoxin B₁ detected previously by ELISA, two additional mycotoxins and one mycotoxin degradation product were detected and quantified by a multi-mycotoxin HPLC-MS/MS approach: Enniatin B and ochratoxin A as well as 2’R-ochratoxin A. Besides the confirmation of co-exposure to several mycotoxins, results probably indicate different exposure routes for the mycotoxins reported.     

Mycoflora,and mycotoxins production in Nigerian corn and corn-based snacks

Thirty-one fungal species, mostly toxigenic and belonging to 11 genera were isolated from corn, corn cake and corn roll snack samples.Aspergillus, Penicillum andFusarium accounted for 10, 6 and 3 of the species and altogether, they constituted 90, 94 and 88 percent of the total fungi in corn, corn cake and corn roll snack respectively. Mycotoxins (aflatoxins and ochratoxin A) were detected in 45, 80 and 12 percent while the means and ranges of the total aflatoxins recorded were: 200(25–770 ppb); 233(15–1070 ppb) and 55(10–160 ppb) for corn, corn cake and corn roll snack samples respectively. Ochratoxin A was detected at toxicologically significant levels in only 15 percent of the corn cake samples analyzed. All the strains ofAspergillus flavus andA. ochraceus tested produced aflatoxin B and ochratoxin A, respectively, when they were cultured on each of the three substrates. In each case, substantial quantities of the toxins were produced from 25 to 35°C with the peak level recorded at 30°C. Toxin production was detected only in substrates with 15 percent moisture content and above; reaching the maximum at 25 or 30 percent moisture level. No substantial differences in the amount of toxins were elaborated with further increase in substrates' moisture content. Of the three substrates, corn cake was the most suitable for aflatoxin B production while they were all equally suitable for the elaboration of ochratoxin A.     

Characterisation of Soybean and Wheat Seeds by Nuclear Magnetic Resonance Spectroscopy

The effects of equilibration under different air relative humidities (RH, 1 – 90 %) and temperatures (35 and 45 °C) on soybean (Glycine max) and wheat (Triticum aestivum) seeds were studied using different techniques. Seed moisture content, electrical conductivity (EC) of seed leachate and per cent seed germination were measured following standard procedures, and compared with nuclear magnetic resonance spin-spin relaxation time (T₂) measurements. Moisture contents of soybean and wheat seeds, following the reverse sigmoidal trend, were greater at 35 than at 45 °C at any particular RH. Changes in T₂ were related to the changes in germination percentage and leachate EC of both soybean and wheat seeds. Equilibrating soybean seeds at RH 11 % decreased germination percentage with corresponding decrease in T₂. On the contrary, EC of seed leachate increased. In wheat seeds equilibrated at 45 °C, T² was maximal at RH 5.5 %. T₂ declined in seeds equilibrated at high RH (> 80 %) together with low germination percentage.     

Solid-Substrate Fermentor for Ochratoxin A Production

A laboratory-scale fermentor designed for solid-substrate fermentation was constructed and tested. Its capacity to produce ochratoxin under varied conditions was determined with wheat as substrate. Ochratoxin yields of 2,000 to 2,500 μg/g of wheat were regularly obtained, and occasionally yields as high as 4,000 μg/g were obtained. The most critical factor in the fermentation was initial substrate moisture content; wheat tempered at 30 to 31% moisture produced the highest yields. Other variables tested were agitation and aeration rates, initial static culture time, and inoculum types and volumes.     

Transformation of the mycotoxin ochratoxin A in plants. 2. Time course and rates of degradation and metabolite production in cell-suspension cultures of different crop plants

Ochratoxin A, one of the most toxic mycotoxins, can be metabolized nearly completely by suspension cultures of various plant cells. The transformation products identified in this study were almost the same in the cell-suspension cultures of maize, carrot, tomato, potato, soybean, wheat and barley, but the quantitative distribution differed strongly depending on incubation time and species of plant-cell culture. The compounds were extracted with ethyl acetate and detected by reversed-phase HPLC with gradient elution. From the result it is supposed that besides ochratoxin A also ochratoxin derivatives may occur in food and feedstuff of plant origin.