International Commission on Food Mycology
International Commision on Food Mycology
Workshop 2010 -“Fungi in food and beverages: new research on spoilage, mycotoxins and prevention”
Freising, Germany,
7 – 9 June, 2010
Abstracts
Session 3: Mycotoxins and Mycotoxigenic Fungi (Naresh Magan)
Influence of osmotic stress on the alternariol biosynthesis in
Alternaria alternata
Eva Graf1*, Markus Schmidt-Heydt1, Christopher Lawrence2, Ha X. Dang2 and Rolf Geisen1
1Max Rubner Institut, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany
2Virginia Bioinformatics Institute, Facility I, Blacksburg, VA 24061-0477, USA
*Presenter: eva.graf@mri.bund.de
Mycotoxin producing Alternaria moulds are ubiquitously present and commonly found on fruits, vegetables, and grains. Some Alternaria species have teratogenic, antibiotic, mutagenic or cytotoxic potential due to their production of mycotoxins such like alternariol or tenuazonic acid. To date, only limited knowledge is available about the regulation of the synthesis of these toxins, especially under food relevant conditions. In foods fungi are exposed to osmotic stress due to a high concentration of different solutes. In the current analysis it could be demonstrated that NaCl shows a deep impact on the alternariol biosynthesis resulting in complete inhibition already at low NaCl concentrations. High osmolarity in the environment is usually transmitted to the transcriptional level of downstream regulated genes by the HOG signal cascade (high osmolarity glycerol cascade), which is a MAP kinase transduction pathway. The inhibition of alternariol biosynthesis by changes in the osmolarity of the substrate might be regulated by this high osmolarity cascade. It therefore became essential to analyse the HOG1 signal transduction pathway of A. alternata in more detail. Expression of the MAP kinase genes hog1 and pbs2 were analysed by real-time PCR. Furthermore, phosphorylation of the HOG1 protein was analysed by western blotting. A clear correlation between HOG1 phosphorylation and alternariol biosynthesis could be established. In addition, homologues of the osmotic pathway of Saccharomyces cerevisiae and different other fungi were aligned with the genomic sequence of Alternaria alternata to get hints for further upstream regulatory genes.
Fungi and toxins in brazil nuts
Beatriz T. Iamanaka1*, Thaiane O. Calderari1, Marta H. Taniwaki1
1Food Technology Institute – ITAL, Campinas, SP, Brazil
Presenter: beatriz@ital.sp.gov.br
Brazil nuts (Bertholletia excelsa) are one of the most important products extracted from the Amazon forest region. One of the major challenges for brazil nut production is to control the high levels of aflatoxigenic fungal contamination and consequent aflatoxin production from these species. The objectives of this study were: (i) to identify the mycobiota of brazil nut kernel and shell; (ii) to evaluate the ability of aflatoxin production by isolates of Aspergillus section flavi; and (iii) to evaluate the levels of aflatoxins in brazil nut kernels. Up to now, 84 samples of brazil nuts (kernel and shell) from different cities of the Amazon region as well as São Paulo markets have been analyzed. The aflatoxin methodology for brazil nut kernels was validated using immunoaffinity columns and HPLC with fluorescence detector. The recovery values for total aflatoxins obtained were: 83.9%, 85.3% and 85.0% for 0.50; 5.0 and 14.6µg/Kg levels, respectively. The detection limit found was 0.03µg/Kg and the quantification limit was 0.2µg/Kg. A total of 7642 fungi were isolated and the infection ranged from 0 to 100% with an average of 83.1%. The main species isolated were: Aspergillus tamarii, Penicillium citrinum, Syncephalastrum racemosum, Aspergillus flavus, Aspergillus section Flavi, Aspergillus section Nigri, Trichoderma spp., Rhizopus spp. and dematiaceos fungi. From the total fungi isolated, 16.7% (1,273 isolates) belonged to section Flavi, including Aspergillus flavus (49.6%), Aspergillus nomius (17.7%) and 32.7% (6 different groups) that are being identified. Regarding A. flavus and A nomius, 27.4% and 100%, respectively were aflatoxin producers. From Aspergillus section Nigri, 6 groups were found, most of which did not produce ochratoxin A and only one group was able to produce this toxin. All of these species isolated from the Amazon region, are being analysed molecularly and from metabolite profiles. Over a half (51%) of the kernel samples showed aflatoxin levels higher than the detection limit. The highest level was found in samples from markets in Para with an average of 7.9mg/Kg.
Ochratoxin A and fumonisin production by Aspergillus section Nigri in food from different origins
Marta H. Taniwaki1*, Beatriz T. Iamanaka1, Larissa Souza Ferranti1, Marina V. Copetti2, Luciana M. R. Esper1
1 Food Technology Institute – ITAL, Campinas, SP, Brazil
2 Federal University of Pampa – Itaqui, RS, Brazil
*Presenter: lumaesper@gmail.com
Knowledge of toxigenic fungi distribution in food is important because it gives parameters to control and prevent mycotoxin production. Ochratoxin A and fumonisin are two mycotoxins produced by Aspergillus section Nigri which are of concern to human health. After a lot of research on coffee, cocoa and dried fruits, several species of Aspergillus section Nigri have been isolated from different origins. The objective of this study was to verify the ability of these isolates to produce ochratoxin A and fumonisins. The isolates were grown in yeast extract 20% Sucrose agar at 25ºC for 7 days. Ochratoxin A was tested from agar plug technique using thin layer chromatography plates under UV light. A total of 408 samples of coffee, 226 of cocoa and 117 dried fruits from all over the world were analysed. From these samples, 1,246 species of Aspergillus section Nigri, were isolated with 88.4% identified as Aspergillus section Nigri, and 11.6% as Aspergillus carbonarius. In spite of this high incidence of Aspergillus section Nigri, only 4.1% were ochratoxin A producers, while 91% of Aspergillus carbonarius produced ochratoxin A. In relation to fumonisin production, so far few isolates from cocoa samples were tested and preliminary results have shown that 87.5% produced fumonisin B2, 25% produced fumonisin B2 and ochratoxin A and 12.5% did not produce fumonisin or ochratoxin A.
Penicillium and Aspergillus species in Australian coffee
Ailsa Hocking1,2* Hayden Druce1 and Peter McGee1
1 School of Biological Sciences, University of Sydney, NSW 2006, Australia
2 CSIRO Food and Nutritional Sciences, North Ryde, NSW 2113, Australia
* Presenter: ailsa.hocking@csiro.au
Coffee has been grown in Australia since the 1880s, but it is only in the past 2-3 decades, with the advent of suitable mechanical harvesters, that coffee growing has re-emerged as a viable industry. Coffee is grown in two main areas, in northern Queensland on the Atherton Tableland at an altitude of 800 – 1100 m, and in the hilly country adjacent to the coast in northern New South Wales and southern Queensland. The Australian coffee industry is very small by world standards, growing only Arabica coffee aimed at the high end of the market. Approximately 750 ha is planted to coffee, producing up to 1600 tonnes of green coffee beans, which is a small fraction of Australia’s annual consumption. No information is available regarding the mycological status of Australian coffee beans.
We sampled beans from two coffee farms in Northern NSW to assess the populations of Penicillium and Aspergillus in the coffee growing environment, in coffee cherries, tree-dried naturals and processed green beans. Penicillium and Aspergillus species were rarely isolated from the surface of green cherries, although a few species were present on tree-dried naturals. In soil, the most commonly encountered Penicillium was P. brevicompactum. From leaf surfaces, P. oxalicum and P. sclerotiorum were isolated. These two species were not encountered on dried coffee cherries or green beans.
Surface-disinfected green beans shelled from tree-dried naturals carried very few fungi. However, in processed green beans, the most frequently isolated fungi were P. brevicompactum, P. citrinum, P. ‘neocrustosum’, P. paxilli and occasionally P. implicatum and P. palitans. Black Aspergillus spp. were relatively abundant, although A. carbonarius was not isolated. Yellow Aspergilli were also common. Of 22 isolates examined, only 5 grew at 37°C, one of which appeared to produce ochratoxin A on coconut cream agar (CCA). Of the 18 isolates that did not grow at 37°C, 12 exhibited blue fluorescence on CCA, indicating OA production.
Almost all Australian coffee is wet processed immediately after harvest then mechanically dried to <0.65 aw. Thus, ochratoxin A contamination is unlikely, even though potentially ochratoxigenic fungi are present on dried green beans.
Ecological comparisons of growth and T-2 and HT-2 toxin production by Fusarium langsethiae strains from northern Europe on an oat-based
medium
Angel Medina1* and Naresh Magan1
1Applied Mycology Group, Cranfield Health, Cranfield , Beds. MK43 0AL, U.K.
Presenter: a.medinavaya.s06@cranfield.ac.uk
Fusarium speciesare important pathogens of cereals and cause significant losses in quality of cereals and concomitant contamination with mycotoxins. Studies in Norway on the occurrence of Fusarium spp. in cereal grain led to the description of a new “powdery Fusarium poae” isolate which produced the mycotoxins T-2 and HT-2. This resulted in the description of a new species, Fusarium langsethiae. This species produces type A trichothecenes, especially the highly toxic T-2 and HT-2 toxins, and diacetoxyscirpenol (DAS). This study has examined the effect of changes in water availability and temperature on both growth and T-2 and HT-2 toxin production by two strains each from Norway, Sweden, Finland and the U.K on a milled oat-based medium. These studies have shown that the temperature range for growth is between 5-30oC with no growth at 37oC. The water activity range varies between strains slightly but generally is limited to about 0.93. The data was used to produce two dimensional contour maps of the optimum and marginal profiles for growth. Statistically, there were very little intra- and inter strain variations in terms of growth.
We examined the effect of these interacting conditions on T-2, HT-2 and the total together. There were significant differences between the ability of strains to produce these toxins and optimum varied between 0.98-0.995 under optimum temperature conditions. Under water stress conditions there appeared to be some changes in the ration of T-2 to HT-2 toxin present in the oat-based cultures. Two dimensional profiles were compared for strains from each country. Studies are in progress to examine the relationship and interaction between the ecology of these fungi and toxin production in the presence and absence of fungicides and studies are in progress on oats.
Acknowledgments:
We are very grateful to Mona Torp, Simon Edwards, Monica Olsen and Marika Jestoi for the supply of the strains.
Chemical and toxicological fate of Fumonisin B1 during extrusion
processing of corn grits
Lauren S. Jackson1, Joseph Jablonski1, Kenneth A. Voss2, Milford A. Hanna3, Andreia Bianchini3, Lloyd B. Bullerman3, and Dojin Ryu4*
1 Center for Food Safety and Nutrition, USFDA, Summit-Argo, IL 60501, USA
2 Toxicology and Mycotoxin Research Unit, USDA-ARS, Athens, GA 30605, USA
3 Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE
68583, USA
4 Department of Nutrition and Food Sciences, Texas Woman’s University, Denton, TX 76204, USA
*Presenter: dryu@mail.twu.edu
Two batches of flaking corn grits were prepared by growing Fusarium verticillioides to contain low and high levels of fumonisin B1 (FB1), Batch-1 at 9.7 ppm and Batch-2 at 50 ppm FB1 as determined by HPLC. These two batches were extruded (Batch-1E; Batch-2E) or extruded with 10% w/w glucose supplementation (Batch-1EG; Batch-2EG) using a twin-screw extruder. FB1 concentrations after extrusion were: Batch-1E = 2.7 ppm; Batch-1EG = 0.6 ppm; Batch-2E = 18 ppm; and Batch-2EG = 5.7 ppm. These values corresponded to FB1 reductions of 72% (Batch-1E), 94 % (Batch-1EG), 64 % (Batch-2E) and 89 % (Batch-2EG). Levels of N-(1-Deoxy-D-fructos-1-yl) FB1 ranged 1.9-2.4 % and 38-46 % of total FB1 species when corn grits were extruded without glucose and with added glucose, respectively. Hydrolyzed fumonisin B1 was a minor species representing <15 % of the total FB1 species present. In toxicological evaluation with rats, relative kidney weights were decreased in groups fed the Batch-1 or Batch-2 diets while apoptotic tubule lesions were found from animals fed the Batch-1, Batch-1E, Batch-2, Batch-2E, or Batch-2EG diets. The severity of the kidney lesions was in correspondence with the FB1 concentration in the diet. Overall, glucose supplementation enhanced FB1 reductions and in vivo toxicity of the extruded corn grits.
Fumonisins from Fusarium and Aspergillus
Ulf Thrane1*, Jesper M. Mogensen1, Thomas O. Larsen1, Maria Månsson1,
Jens C. Frisvad1, Kristian F. Nielsen1
Center for Microbial Biotechnology, Department of Systems Biology,
Technical University of Denmark, Søltofts Plads 221, DK-2800 Kgs. Lyngby, Denmark
*Presenter: ut@bio.dtu.dk
A group of economically important mycotoxins, the fumonisins, was for many years considered to originate from only a few Fusarium species. However, the sequencing of the entire genome of Aspergillus niger revealed that also this species has the full gene cluster for fumonisin production, and it has been demonstrated that A. niger can produce fumonisins. For analysis of fumonisins in pure cultures a simple agar plug extraction technique has been developed. After extraction of agar plugs with 75% methanol in water the extracts are filtered and analysed for fumonisins by LC-MS. It has been shown that A. niger has a specific fumonisin profile of B2, B4 and a new fumonisin, B6. The latter has so far not been found in any Fusarium culture. Our results show that the production of fumonisins is regulated differently in Fusarium and A. niger species, as Fusarium requires high water activity and higher (neutral) pH for fumonisin production, whereas media with 10-20% sucrose, 2.5-5% NaCl or lowered pH increased the production of fumonisins by A. niger. Fusarium cultures have the maximal production of FB1 and FB2 at 20-25°C, while 25-30°C is optimal for A. niger cultures. To some extent these observations fit very well to the ecological niches where Fusarium and A. niger, respectively, may grow in nature, but our observations also show that even if the gene cluster for a well-known mycotoxin is fully sequenced, it is not possible to predict which ecological conditions may express the genes leading to mycotoxin production. This highlights the importance of knowledge on the pro- duct associated mycobiota, i.e. knowledge on which fungal species can grow on food and feed since they might produce mycotoxins.
Fumonisins from Aspergillus in Foods
Jens C. Frisvad1*, Jesper M. Mogensen1, Thomas O. Larsen1, Marie Sørensen1, Robert A. Samson2, Ulf Thrane1 and Kristian F. Nielsen1
1Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Building 221, Søltofts Plads, DK-2800 Kgs. Lyngby, Denmark
2CBS Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD Utrecht, the Netherlands
*Presenter: jcf@bio.dtu.dk
Apart from Fusarium spp., one species of Aspergillus is able to produce fumonisins: Aspergillus niger. Other species in Aspergillus section Nigri have not been shown to be able to produce fumonisins. A large percentage (approximately 80%) of isolates of A. niger are able to produce fumonisins B2, B4 and B6, while only 5-10% can produce another potentially carcinogenic mycotoxin: ochratoxin A. In contrast to fumonisin production by Fusarium spp., A. niger produces large amounts of fumonisins on media with a high concentration of sugars or salt. Synergy in stimulation of fumonisin production by two carbon sources was shown with lactate and starch, indicating that replacing fat with starch in low fat products may be risky, if Aspergillus niger is a contaminant of, for example, salami. Fumonisin B2 and B4, produced by A. niger, have been found in wine, coffee, and artificially inoculated grapes and raisins, but A. niger can grow in many foods and feeds. Until recently maize was often the only important food that was examined for fumonisins, because Fusarium verticillioides was associated to this crop, but with the knowledge that A. niger can produce fumonisins, many kinds of foods should be examined for this mycotoxin. Confirmed reports of fumonisin B2 presence in rye bread and figs, for example, indicated that those substrates were contaminated by A. niger rather than Fusarium species, as the latter produce more fumonisin B1 than B2. Other substrates where A. niger may produce black rot, such as onions, pepper and mango, should also be examined for fumonisins. Puerh tea and black tea are also often contaminated by black Aspergilli, but seems to be contaminated by preferentially Aspergillus acidus, and this species appears to be a non-producer of fumonisins. Black Aspergillus species can be selected by 20% tannic acid, but not on agar media, as the agar cannot settle in such acidic media. Furthermore there are no direct methods to select A. niger from other black Aspergilli, and a polyphasic identification is recommended.
