Unraveling the degradation pathway of zearalenone (and aflatoxins) by Actinobacteria using a poly-omics approach
Promotor: Leen De Gelder, Kris Audenaert, Filip Van Nieuwerburgh (UGent) Researcher: Laura De Mets (UGent) Period: 01/2017-12/2020
Mycotoxins and mycotoxin contaminated feed and food are continuing to be a worldwide problem. One of the most recent strategies to combat this problem, is the microbial detoxification of mycotoxins. A poly-omics approach will be used to fully explore the mycotoxin-degradation potential of Actinobacteria such as Streptomyces sp. and Rhodococcus sp., which are known for their catabolic versatility. By using a poly-omics approach, including genomic and transcriptomic analysis, we aim to completely unravel the degradation pathway of zearalenone. This will allow us to pinpoint important genes, enzymes and degradation steps: a gateway towards practical application of the remediation strategy.
Interaction between Fusarium mycotoxins and cytochrome P450 drug metabolizing enzymes/ABC drug transporters in a porcine animal model
Induction or inhibition of cytochrome P450-drug metabolizing enzymes and ABC transporter proteins by co-administered drugs or food has been associated with a number of clinically significant drug-drug or drug-food interactions in humans. Concerning food chemical contaminants, mycotoxins are highly prevalent fungal metabolites and have shown to be the number one threat regarding chronic toxicity to humans and animals. Most drug-drug or drug-food interactions at the level of the human intestinal mucosa are studied in vitro using cell culture models and rodent intestinal tissue. A primary concern for in vivo studies, is the lack of a representative animal model which estimates the impact of these interactions on the in vivo pharmacokinetic behavior of substrate drugs in humans. Pigs may be introduced as animal model in the safety assessment of pharmaceutical or chemical products because of the striking anatomical, physiological and biochemical resemblances between humans and pigs. This research proposal is intended to contribute to the development of such a porcine model, including the characterization of porcine metabolic enzymes and drug transporters, their modulation by frequently detected mycotoxins in food and the impact on the pharmacokinetic disposition of substrate drugs.
The further outcome of this project will help in understanding and predicting drug-drug and drug-food interactions using a porcine model, leading to improved drug safety and effective drug therapies.
FUSARIUM MYCOTOXINS AND THEIR MODIFIED FORMS IN NIGERIAN FOODS: OCCURRENCE AND INFLUENCE OF TRADITIONAL PROCESSING METHODS
Promotor: Sarah De Saeger (UGent), Marthe De Boevre (UGent) and Olusegun Atanda (MCU Nigeria) Researchers: Cynthia Adaku Chilaka (UGent) Period: 2015-2018
Fusarium is one of the most common and important filamentous pathogenic fungi widely distributed around the world. The fungus can contaminate a wide variety of crops in the field and during storage. In addition to causing economic losses, Fusarium spp. synthesizes under certain conditions various mycotoxins with subsequent toxic properties. Studies have demonstrated that metabolization of mycotoxins in planta by biological modification or chemical modification through transformation during food processing or interaction of mycotoxins with matrix compounds can lead to the formation of conjugated or modified toxins referred to as modified mycotoxins. Modified mycotoxins when ingested might subsequently hydrolyse to the parent toxins in the digestive tract of the organisms thus causing additional health effect to the host. Limited research on occurrence of Fusarium mycotoxins and their modified forms in food has been reported in the African continent especially Nigeria.
This project aims to investigate the occurrence of Fusarium mycotoxins and their modified forms in Nigeria staple crops, as well as their locally processed food products. The fate and stability of Fusarium mycotoxins during traditional processing of fermented Nigerian foods will also be investigated.
The incidence of citrinin in the Belgian food and feed chain and the risk for human and animal health
Promotor: Sarah De Saeger (UGent), Siska Croubels (UGent) and Mathias Devreese (UGent) Researchers: Celine Meerpoel (UGent) and José Diana Di Mavungu (UGent) Period: 10/2016-10/2020
Within this research project, the research partners will gather information on the occurrence of citrinin in feed and different foodstuff available on the Belgian market with focus on the discovery of all relevant sources of intake and their importance. (U)HPLC-MS/MS methods to determine citrinin in feed, different food groups and different edible tissues of animal origin will be optimized and validated. Furthermore, the toxicokinetics and absolute oral bioavailability for citrinin in broiler chickens and pigs, and carry-over to edible tissues (kidney and muscular tissue from pigs and broiler chickens; eggs from laying hens) will be investigated. The pig may also serve as toxicokinetic model for humans, given the anatomical and physiological similarity of inter alia the gastro-intestinal tract, liver and kidneys. Additionally, the toxicity and organ damage in pigs, broiler chickens and laying hens will be investigated. Currently, one phase I metabolite of citrinin has already been described, i.e. dihydrocitrinone, but the use of high resolution mass spectrometry (HRMS) will enable us to explore other potential phase I and II metabolites to unravel its biotransformation. The reference materials for citrinin already developed within the CEN-project (CODA-CERVA), will be placed at the disposal of the research partners. In the end this research project will perform a risk assessment for citrinin in Belgium.
Biological control of Fusarium graminearum with fungal and bacterial antagonists on crop and crop residues
Promotor: Sarah De Saeger (UGent), Geert Haesaert (UGent), Kris Audenaert (UGent) Researcher: Mohamed Fathi Abdallah (UGent) Period: 08/2016-08/2019
Fusarium Head Blight (FHB), a devastating fungal disease of several cereals including wheat, is caused by Fusarium graminearum (Fg). The disease affects the wheat grain both qualitatively and quantitatively. Beside the economic losses, the fungus produces secondary toxic metabolites, called mycotoxins. These metabolites have a serious impact on animal and human health upon the consumption of contaminated wheat. The main source of Fg inoculum for wheat are crop residues. Overwintering and sporulation on these residues are responsible for most Fusarium spp. infections, and subsequent mycotoxin contamination. Conservation tillage practices maintain soil fertility and prevent erosion, while leaving more stubble/straw residues and weeds which can feed the primary inoculum of Fg. Even after plowing, a limited quantity of maize stubble is an important source for FHB infection. Biological control is a safer and cheaper solution for fungal disease management in comparison to other chemical fungicides.
The main aims of this project are:
To select and formulate (an) efficacious fungal and bacterial antagonistic strain(s) to be applied onto the preceding crop (endophytic growth and/or crop residues) before maize and wheat to reduce Fusarium inoculums.
To enhance the efficacy of this approach by better understanding the interaction between applied antagonists, Fusarium species and natural occurring micro-organisms.
To investigate the effect of the biocontrol agent on the secondary metabolism of the pathogen.
The current project is a part of the MYCOKEY project that aims at ‘Integrated and innovative key actions for mycotoxin management in the food and feed chain’. The project is funded by Horizon 2020 and includes 32 partners from the EU, China, Argentina and Nigeria.
Toxicokinetic study of the modified mycotoxins deoxynivalenol-3-glucoside, zearalenone-14-glucoside and zearalenone-14-sulfate in the juvenile pig as human paediatric surrogate model
Promotor: Siska Croubels (UGent), Mathias Devreese (UGent), Sarah De Saeger (UGent) Researcher: Amelie Catteuw (UGent) Period: 01/2017-01/2021
Additionally to the highly hazardous free Fusarium mycotoxins DON and ZEN, modified forms frequently occur in food and feed as well. They are produced by plants and rival fungi by altering the chemical structure and hereby reducing the toxicity of the native mycotoxin as part of a natural defense strategy against xenobiotic compounds. Important examples of these modified mycotoxins are deoxynivalenol-3-glucoside (DON3G, conjugated by plants), zearalenone-14-glucoside (ZEN14G, conjugated by plants and fungi) and zearalenone-14-sulfate (ZEN14S, conjugated mainly by fungi). Relative high incidence rates have been noted for given modified mycotoxins in food and feed.
The available in vitro and in vivo studies focusing on modified mycotoxin’s toxicokinetics and –dynamics (TK/TD) characteristics demonstrate the toxic relevance of these forms, as modified mycotoxins can be cleaved (hydrolysed) during mammalian digestion, releasing the free mycotoxins. However, little is known about TK/TD properties of DON3G, ZEN14G and ZEN14S in both young animals and in infants and young children, a vulnerable population group. The general objective of this research is to determine in vitro intestinal cytotoxicity and in vivo disposition, i.e. oral bioavailability, rate and extent of hydrolysis and toxicokinetic characteristics of DON3G, ZEN14G and ZEN14S in the juvenile pig.
The juvenile pig is a relevant animal species, firstly because of its high exposure and sensitivity to mycotoxins and consequently economic relevance, and secondly because of the possible extrapolation to infants and young children, and consequently public health relevance.
Results of this project will support legislative authorities to carefully consider specific aspects of the risk assessment and establishment of maximum levels of DON3G, ZEN14G and ZEN14S in feed and foodstuff as hazard to infants and young children, and will help farmers and veterinarians to support the diagnosis of mycotoxin exposure.
Interactions between mycotoxins and the rumen, their possible toxicological effects on the gastrointestinal tract and intestinal absorption in dairy cattle: an in vitro approach
Mycotoxins are more and more associated with subclinical health problems for high productive dairy cows reflected by vague and non-specific symptoms and periodic decrease in milk production. Indeed, the risk of mycotoxin contamination of dairy diets is high since the main components such as corn, grass silage and small grain cereals are susceptible to infection with toxigenic fungi. Moreover, considering the wide diversity of toxigenic fungal species on crops and the ability of several fungi to produce more than one mycotoxin, a multiple contamination can be expected. Hence, dairy cows are exposed to various mycotoxins which may lead to depletion of the detoxifying capacity of the microbiota in the rumen. To date more and more dairy farmers and veterinarians are concerned about the impact of mycotoxins on the health and performance of dairy cows. Therefore, research about this topic is needed.
This multidisciplinary doctoral research will elucidate the degradation of individual mycotoxins and relevant mycotoxin combinations in the rumen, the interactions between mycotoxins and the rumen function as well as the possible toxicological effects of mycotoxins on the gastrointestinal tract and their intestinal absorption. Mycotoxins that frequently contaminate corn silage, the most important part of the diets of dairy cattle, will be studied by means of in vitro rumen simulations. Also varying rumen conditions will be included (e.g. varying dietary ratios roughage/concentrate up to circumstances of subacute acidosis), as they may influence mycotoxin degradation. UHPLC-MS/MS (Xevo TQ-S) and UHPLC-HR-MS (Synapt G2-Si HDMS) instruments will be applied to identify and quantify mycotoxins and their major metabolites in the rumen contents. Intestinal cytotoxicity and absorption of mycotoxins will be determined by means of relevant cell cultures. In addition, the efficacy of mycotoxin binders will be tested at the level of rumen metabolism, cytotoxicity and intestinal absorption.
This doctoral research is part of an IWT LA project “Development of a decision support system for better control of mycotoxins in maize silages; LA 140971”.
A decision support system to control mycotoxin contamination in maize silages
Mycotoxins are toxic secondary metabolites produced by a variety of fungal species, such as Fusarium, Penicillium or Aspergillus, among others. Contamination of feed with mycotoxins can cause severe health problems in dairy cattle. Especially high yielding dairy cows with a high feed uptake and rapid ruminal flow are susceptible to gastroenteritis, reduced reproduction and reduced milk production, as a result of mycotoxin contamination.
Maize silage is one of the main components of dairy feed in the region of Flanders, Belgium, and is therefore one of the main sources for mycotoxin uptake in dairy cows. This research aims towards providing dairy farmers in Flanders with a user-friendly prediction model, able to foresee mycotoxin contamination based on weather, cultivation, harvest and silage conditions.
This model will be constructed based on analyses of maize silages across Flanders, and on own research focusing on methods to prevent mycotoxin contamination. 100 maize silages will be selected based upon geographical spread, cultivation technique and silage conditions. These silages will be sampled once during harvest and 2-3 times during feeding every year for four years, and analyzed for mycotoxin and fungal contamination. Own research will be divided into five separate work packages, with the following topics: biofumigation of the soil using green crop manures, treatment of crop residues with antagonistic microbial populations, impact of harvest date and dry matter content on mycotoxin contamination, microbial detoxification in the silage, and toxicity of mycotoxins in dairy cattle. These results will aid towards constructing and validating the prediction model.
Microbial degradation of the mycotoxin deoxynivalenol
Promotor: Leen De Gelder (UGent), Kris Audenaert (UGent), Sarah De Saeger (UGent) Researcher: Ing. Ilse Vanhoutte Period: 1/10/2015-30/09/2020
Contamination of food and feed by mycotoxins, poses major risks for animal or human health and leads to economic losses. Next to prevention and intervention measures on the field, remediation of mycotoxins in contaminated feed is a growing sector. Remediation is already applied through the use of binders (clay or yeast derived products), but this adsorption between the binder and mycotoxin is reversible and pH depending. Moreover, these binders negatively influence the transfer of medication to the bloodstream. Therefore, there is need to develop other detoxification strategies. This can be achieved through microbial degradation of mycotoxins. Matrices with potential levels of mycotoxins or other complex molecules are screened on the presence of microorganisms which can break down DON. Enrichment cultures for DON at 50 mg/kg have been established for the matrices: sheep rumen fluid, monoculture corn field soil, digestate from an anaerobic digester plant and activated sludge from a water treatment plant. Clear detoxification of DON by the enrichment cultures of soil and activated sludge was assessed with a bio-assay using Lemna minor (duckweed), whereas the actual degradation of DON through these cultures was confirmed with ELISA and LC-MS/MS analysis.
Using the plant volatile arsenal to combat fungal contamination in crops
On a European level, the use of pesticides and fungicides is increasingly discouraged, while the implementation of Integrated Pest Management is encouraged. In this framework, the search for biostimulants, which have a protecting effect on plant health against (toxin-producing) pathogens, is gaining traction.
Plants release a plethora of Biogenic Volatile Organic Compounds (BVOC) in response to both abiotic and biotic stress, a large group within the BVOC bouquet are the Green Leaf Volatiles (GLVs). It derives its name from the fragrance which is typically associated with freshly cut grass. GLVs are implicated in enhancing defense against insect herbivory. However, we have found that GLVs also offer a protection against fungal infection.
This research project aims at uncovering the potential of GLVs as a crop protection agent against different mycotoxin producing fungi (Fusarium spp., Cochliobolus spp. etc.). We will focus on the effect of GLVs on both plant disease and mycotoxin production as these are known to have a large influence on each other.
CHARACTERIZATION OF ALTERNARIA SPECIES ON FLEMISH POTATO
Promotor: Kris Audenaert (UGent), Monica Höfte (UGent), Sarah De Saeger (UGent) and Geert Haesaert (UGent) Researcher: Michiel Vandecasteele (UGent) Period: 2013-2017
Alternaria species, including A. solani and A. alternata are a serious threat for potato cultivation since heavy infections can lead to significant yield and quality losses. Both species cause necrotic symptoms, which cannot be visually distinguished. Over the past years, both pathogens have become increasingly important in NW Europe. Although the exact cause for this emerging problem remains elusive, it might be attributed to the combined effect of climate change, a reduced use of the fungicide mancozeb, the increased specificity of active ingredients to control Phytophtora infestans and the production of high-yield susceptible cultivars. Furthermore, little is known about the Flemish Alternaria population and about the contribution of both species to the disease. The main goal of this research is to identify the primary causal agent of potato early blight, to determine inter- and intraspecific diversity within the Alternaria population in Flanders, and to unravel the complex interaction between stress-related hormones and the Alternaria infection.
Intestinal toxicity and oral resorption of conjugated Fusarium mycotoxins, frequently found in food & feed
Promotor: Siska Croubels (UGent), Mathias Devreese (UGent), Sarah De Saeger (UGent) Researcher: Nathan Broekaert (UGent) Period: 2012-2015
Additional to native mycotoxins, conjugated mycotoxins can also be found in contaminated food and feed. These conjugated forms are also known as “masked mycotoxins” due to the fact that they often remain undetected by standard analysis methods and routine controls. The absence of commercial analytical standards of these compounds does not ameliorate the situation. These conjugated forms may be produced by plants (e.g. glycosylated derivates), by the fungus itself (e.g. acetylated derivates) or during food processing. A literature review indicates that these masked mycotoxins can reach high levels that can even exceed these of the native toxins. As there is no data available in man or animal on the biological availability and intrinsic toxicity of these compounds it is important to obtain some insights into the matter.
Within this project the first focus will lay on the synthesis and isolation of glycosylated derivates of deoxynivalenol (DON) and zearalenone (ZEN) as well as acetylated DON derivates, and this in amounts sufficient to conduct further in vitro and in vivo trials.
These synthesized masked mycotoxins will be utilized to determine the intestinal cytotoxicity and the influence on the cytokine response of these compounds. In a further stage, the oral bioavailability and in vivo degree of hydrolysis will be determined of these conjugated toxins by using in vitro en in vivo toxicokinetic models. Pigs and chickens will serve as target animals as these animals have the highest natural exposure to these toxins and are economically relevant. Additionally, pigs can serve as a model for human toxicokinetics.
The research will generate data concerning the toxicokinetic en toxicological properties of masked mycotoxins. Based on these properties one can make a more informed decision on the potential need to include these compounds in routine controls, and possibly legislation.
(Masked) Alternaria mycotoxins in foodstuffs: Occurrence and influence of food processing
Promotor: Sarah De Saeger (UGent), Lynn Vanhaecke (UGent) and Anita Van Landschoot (HoGent)
Researchers: Jeroen Walravens (UGent) and José Diana Di Mavungu (UGent)
Period: 01/01/2013 – 31/12/2015 (ALTER – RT 12/6261 – FBS Health, Food Chain Safety and Environment
Alternaria is a common genus of fungi, which contains numerous species that can contaminate a wide variety of crops in the field and cause post-harvest decay of various fruits, grains and vegetables. In addition to causing economic losses, Alternaria spp. form under certain conditions mycotoxins with toxic properties. There might be a possible risk for public health related to the presence of Alternaria toxins in feed and food. Additionally, mycotoxins can, as other xenobiotics, partly be metabolised, e.g. the formation of conjugated Alternaria toxins in plants. The presence of these “masked” mycotoxins can lead to an underestimation of the mycotoxin contamination in foodstuffs. Recently, the European Food Safety Agency (EFSA) stipulated that additional quantitative occurrence data on Alternaria toxins need to be gathered. Additionally, only scarce information on the stability and fate of Alternaria toxins during storage and processing of food is available. Through the development and validation of a multimycotoxin analytical method, this research project will gather information on the occurrence of Alternaria mycotoxins in cereal products, tomato products, fruit and vegetable juices and beer. Additionally, the effect of food processsing on the toxin levels will be investigated, during the beer production chain and the production of tomato based foodstuffs (juice, sauce and concentrates). The fact that not only the native Alternaria precursors, but also their masked metabolites will be investigated, highlights the innovative aspect of this research project.
Mycotoxins and mycotoxigenic fungi in China: analytical tools, dietary exposure and Fusarium diversity
Promotor: Sarah De Saeger (UGent)
Belgian partners: Stéphane Declerck (Université catholique de Louvain, UCL), Alfons Callebaut (CODA-CERVA), Geert Haesaert (University College Ghent, Hogent)
Chinese partners: Dr.Y.Qi (Chinese Academy of Tropical Agricultural Sciences, CATAS), Prof. Y.-C. Liao (Huazhong Agricultural University, HZAU), S. Peng (Shanghai Food and Drug Administration, SHFDA), H. Gao (Shanghai Food and Drug Administration, SHFDA), Prof. J. Shen (China Agricultural University, CAU), Prof. S. Zhang (China Agricultural University, CAU), Prof. D. Zhang (Shanghai Jiao Tong University, SJTU), Dr. A. Wu (Shanghai Academy of Agricultural Sciences, SAAS)
Researchers: J. Diana Di Mavungu (Ghent University, UGent), N. Beloglazova (Ghent University, UGent), F. Munaut (Université catholique de Louvain, UCL), F. Van Hove (Université catholique de Louvain, UCL), Kris Audenaert (University College Ghent, Hogent), B. Huybrechts (CODA-CERVA), E. Tangni (CODA-CERVA), Ph. Debongnie (CODA-CERVA), H.-P. Li (Huazhong Agricultural University, HZAU), J.-B. Zhang (Huazhong Agricultural University, HZAU), J.-H.Wang (Huazhong Agricultural University, HZAU), Z. Wang (China Agricultural University, CAU)
The major aim of this proposal is to bring together experts from both China and Belgium to conduct research on mycotoxins and mycotoxigenic fungi, including Fusarium and related toxins. The different partners will be complementary as they are focused on different aspects of this research topic. Multidisciplinarity and cross-border research are the key issues in this project.
More specifically, the project will have the following objectives:
Development of analytical tools to assess mycotoxin dietary exposure in China.
Study on the genetic and mycotoxigenic diversity of Fusarium on wheat, maize and banana in China.
Assessment of mycotoxin exposure in the Belgian population using biomarkers
Coordinator: Sarah De Saeger (UGent)
Promotors: Stefaan De Henauw (UGent) and Alfons Callebaut (Coda-Cerva)
Researchers: Ellen Heyndrickx (UGent), Isabelle Sioen (UGent) and José Diana Di Mavungu (UGent)
Period: 01/10/2011 – 30/09/2014; Project RT 11/02 BIOMYCO FBS Health, Food Chain Safety and Environment
Mycotoxins are one of the most important chronic risk factors in our food chain, therefore it is important to estimate the exposure of the population to these toxins. Currently, the assessment of mycotoxin exposure is based on the occurrence in food and questionnaire data on food consumption. This indirect approach is not reliable due to the inaccuracy of food consumption data, the heterogeneous distribution in food, the exposure through inhalation and the presence of masked mycotoxins. A more accurate assessment of the exposure can be performed by direct measurements of biomarkers of exposure.
The objectives of this project are:
Measurement of mycotoxins via biomarkers in urine taken in samples of a representative part of the Belgian population, using validated analytical methods
Perform an exposure assessment via biomarkers for a number of mycotoxins to which the Belgian population is exposed (mainly through dietary intake)
Investigate the correlation between the measured biomarkers and the reported consumption of different groups of food commodities
MYTOXPLEX: Is the occurrence of the T2/HT-2 mycotoxin complex in cereals a new and actual problem in the food and feed chain?
Promotors: Geert Haesaert (HoGent), Kris Audenaert (HoGent), Sarah De Saeger (Ugent), Françoise Munaut (UCL) and Mia Eeckhout (HoGent)
This project will use an explorative approach to:
Assemble qualitative and quantitative T2 and HT-2 data in small grain cereals (wheat, barley, oat, spelt and triticale) and derived cereal products produced and handled in Belgium.
Elucidate the occurrence of T2 and HT-2 producing Fusarium species and their genetic diversity in order to come up with appropriate control measures.
Identify the presence of other mycotoxins produced by F. poae and F. langsethiae.
Assess the importance of other Fusarium species within the T2/HT-2 complex.
Implement a preliminary risk assessment for T2, HT-2 and other eventual mycotoxins within the F. poae group based on an exposure estimation.
Alternaria spp. on potato: genetic characterization, chemotyping and disease development in relation to abiotic stress
Promotors: Geert Haesaert (HoGnt), Kris Audenaert (HoGent), Sarah De Saeger (UGent), Monica Höfte (HoGent)
This project aims to characterize the population of Alternaria and to decipher the role of abiotic stress in the infection and colonization process in potato. A more thorough insight into the complexity of the pathogen will result in a better control with reduced use of fungicides. Several research questions will be addressed:
How complex is the Alternaria population in the Flemish Potato fields?
What are the positions of Alternaria alternata and Alternaria solani within the disease complex? Is A. alternata a weak pathogen solely infecting wounded tissue or is it a true pathogen?
What is the intra-specific genetic diversity of the population and how does this affect virulence?
Are abiotic stress factors inherent to the potato plant a trigger for the colonization of Alternaria? Is there a role for mycotoxins and how do they interact with abiotic stress hormones like ethylene?
How can this project result in a better control of the pathogen complex in the field?
Researching Fusarium poae: its mycotoxines, chemotype and influence of oxidative stress triggers
Fusarium head blight (FHB) is a fungal disease that can occur on several cereals, including wheat. An important characteristic of FHB is that the disease is caused by a blend of up to 17 distinct species causing identical symptoms. The interest in FHB research is primarily fueled by the potential of FHB pathogens to produce mycotoxins. These mycotoxins are low-molecular organic compounds that can be toxic for humans and animals and as such implicate a serious health issue.
Thorough research at the Ghent University College recently attempted to shed light on the ever changing composition of the Fusarium complex. It was found that in Flanders, Fusarium poae has an important place within this disease complex. Up to recently, research in this field did not focus on F. poae, as other species were much more aggressive within the complex. Its rising presence, its nature as a secondary attacker and its potential to produce mycotoxins which could be much more toxic than those of other species, illustrate the large need for research on this topic.
The Fusarium poae research at the Ghent University College aspires to fill the gaps in the scientific community’s knowledge on this fungus. Field isolates that originate from several testing locations in Belgium are characterized pursuing a multidisciplinary approach of chromatographic, genetic and pathological methods. LC-MS/MS techniques are used to unravel the chemotype of the isolates. To this end, the Fusarium poae project has ties with the ‘Mytox’ Association Research Group (AOG), and research is carried out in the Bromatology lab at the Faculty of Farmaceutical Sciences at the University Ghent. The qualitative and quantitative production of toxins is examined under different conditions and influences of stress, such as fungicide treatment.
A further characterization of the field samples involves fungicide resistance assays of F. poae and the related species F. langsethiae to fungicides such as triazoles and strobilurines. Experiments to examine the physiological effects of fungicide treatments on both a macroscopic and a microscopic level are set up. This work is carried out in the broader framework of oxidative stress as a trigger for toxin production, which could be a central dogma in toxin production in all toxigenic fungi. To find a measure for aggressivity of the isolates, detached leaf assays are used. This technique also allows us to test several sources of resistance in wheat for its strength against F. poae. Again, microscopy visualization techniques are employed, this time to gain insight in the infection strategy of the fungus and the plant’s corresponding response. The last piece of the puzzle is filled in when AFLP-fingerprinting lets us unravel the genotype of the different isolates.
These different approaches of the Fusarium poae problem allow us to paint a fairly complete picture of the fungus, and as such meet the scientific community’s need for more knowledge on this organism with its importance increasing now more than ever.
SHORT TRAINING INITIATIVE
The Short Training Initiative (STI – VLIR-UOS) with as promoter Prof. dr. Ph. Sarah De Saeger is an intensive training on mycotoxin analysis. The main target group are researchers from developing countries working for the government, universities and NGOs or students from developing countries at the final year of their Masters degree program. This 9-day training is planned for the end of August, beginning September 2012.
Mycotoxin contaminations occur world-wide and represent one of the most challenging and prominent food safety threats. In terms of exposure and severity of chronic disease, especially cancer, mycotoxins appear at present to pose a higher risk than anthropogenic contaminants and pesticides. They seriously affect the agricultural economies of many countries, interfere or even prevent trade, reduce animal and animal product production and affect human health. Between 25% and 50% of all commodities, especially staple crops, are contaminated with mycotoxins. In developing countries, monitoring and enforcement (according to EU or US legislation) only occurs occasionally when commodities such as groundnuts and coffee are destined for export while it is mostly ignored for local goods. Agricultural commodities of inferior quality are kept for local use resulting in adverse and even fatal health effects. Fortunately, the awareness on the mycotoxin problem is growing in several developing countries.
This training session aims teaching the trainees most important analytical methodologies for mycotoxin analysis in food and feed. This will be done by practical exercises at the Laboratory of Food Analysis, Ghent University, Belgium.
Moreover, the awareness on the mycotoxin problem in developing countries will be further enhanced by providing the trainees with theoretical lessons given by different experts on the several aspects of the mycotoxin issue.
MycoHunt: Rapid biosensor for the detection of mycotoxin in wheat
Promotor: Sarah De Saeger (coordinator UGent), Mia Eeckhout (coordinator HoGent)
Collaborator: Yirong Guo (UGent), Melanie Sanders (UGent), Freya Martens (HoGent)
MycoHunt is a project granted in the EU7 framework program for the development of a rapid online biosensor for the detection of mycotoxin in wheat.
The project aims to increase the competitiveness of a large group of “Small and Medium Enterprises (SME) Associations” in Europe by developing a cost-effective method to detect the contamination of deoxynivalenol (DON) in wheat grains. This mycotoxin forms a major threat in the food and feed sector of the European industry. A group of SME Associations, covering the relevant sectors and representing a vast number of sector SMEs, participate in the project. In this way, they will gain knowledge and resources to further exploit the results of the novel technology developed by providing a thorough sampling and measurement method of grain.
In the framework of the project the development of an online, non-destructive sampling apparatus for dust and other low molecular weight particles is targeted. Another aspect of the research is the development of a specific sensor based on an immunoassay method using DON specific monoclonal antibodies. Beside the technological objectives, the research focuses on the determination of parameters (temperature, pressure, vacuum, etc.) affecting the sampling precision. Also different immobilizing methods for the DON specific monoclonal antibodies will be investigated. The determination of the cross-reactivity of these monoclonal antibodies against other trichothecenes, like 3-acetyl-DON, 15-acetyl-DON, nivalenol, is also an interesting point to consider.
The research leading to these results has received funding from the European Community’s Seventh Framework Program (FP7/2007-2013) under grant agreement n°243633.
Development and application of molecularly imprinted polymers (MIP’s) in mycotoxin diagnostics
coordinators: Nicolas Gryson (HoGent) and Peter Maene (HoGent)
Copromoters: Sarah De Saeger (Ugent) and Peter Dubruel (UGent)
Collaborators: Frederic Dumoulin (UGent)
Fumonisin (FUM) is a mycotoxin that is mainly present in corn and derived products. The presence of fumonisin in food and feed products may pose a risk to human and animal health.
Current detection methods for fumonisins are based on the use of immuno-affinity columns. Due to several disadvantages related to the development (use of laboratory animals, long development stage) and the use of these columns (high cost, single use), there is an increased demand for alternative user-friendly detection methods.
The goal of this research project is to develop a quick test for the detection of fumonisin in corn products with the aid of molecularly imprinted polymers (MIPs).
A first step involves the development and characterisation of the specific MIP. Main variables in this process are the choice of the functional monomer molecule, the cross-linker molecule and the porogeneous solvent used during polymerisation. The characterisation basically implies the determination of the binding capacity. In a second step, the procedure for fumonisin extraction from different corn products will be optimised, using a solid-phase extraction method. The optimised method should guarantee a recovery of 70 to 110 %. Based on those two steps, a quick test for fumonisin detection will be developed. This is a semi-quantitative test based on fluorescence detection. The developed test will be an adequate tool for food and feed companies for the auto control of samples, in order to comply with the current Belgian and European legislation on food and feed safety. The test will be fast, simple, cheap and user-friendly. It will therefore be a convenient tool for all stakeholders in the corn supply chain in the prevention of fumonisin contamination.
Investigation of new analytical challenges for mycotoxin determination: Masked mycotoxins
Promoter: Carlos Van Peteghem (UGent)
Copromoters: Sarah De Saeger (UGent), Dieter Deforce (UGent), Geert Haesaert (HoGent), Mia Eeckhout (HoGent)
Researchers: Marthe De Boevre (UGent), José Diana Di Mavungu (UGent), Peter Maene (HoGent)
Period: 01/08/2009 – 31/07/2013
Project number: BOF 05V00709, Research association project
Conjugated mycotoxins, in which the toxin is usually bound to a more polar substance such as glucose, are referred to as masked mycotoxins, as these substances escape established anlatytical techniques but can release their toxic precursors after hydrolysis. Little is known about the occurrence of these compounds in foodstuffs.
High-performance liquid chromatography (HPLC) combined with tandem mass spectrometry (MS/MS) offers a powerful tool for identification and characterization of mycotoxin conjugates. Derivatives can be identified due to their molecular mass and their collision induced dissociation (CID) fragmentation behavior, and unknown mycotoxin conjugates can also be identified with different MS techniques, namely quadrupole time-of-flight (Q-ToF) MS and Fourier transform (FT) MS.
The aim of this project is to evaluate the applicability of LC-MS/MS en high resolution MS tackle the problem of masked mycotoxins. Through this research project, an answer to the following questions will be formulated:
Which mycotoxin conjugates are found in cereal-based food and feed?
Is the occurrence of masked mycotoxins dependent on the cereal variety and genotype?
When and how are conjugated mycotoxins formed?
What is the share of masked mycotoxins to the total mycotoxin content?
Do food processing technologies play a role in the occurrence of masked mycotoxins?
Do mycotoxin conjugates contribute to overestimations of mycotoxin levels with commercial ELISA kits as compared to the traditional LC methods?
Assessment of the chemical risk associated with masked Fusarium toxins
Coordinator: Sarah De Saeger (UGent)
Copromoters: Carlos Van Peteghem (UgGent), Mia Eeckhout (HoGent), Geert Haesaert (HoGent), Kris Audenaert (HoGent)
Researchers: Marthe De Boevre (UGent), José Diana Di Mavungu (UGent), Peter Maene (HoGent)
Conjugated or masked mycotoxins first came to attention in the mid-1980s, when animals fed with apparently low mycotoxin contaminated feed showed high severity of mycotoxicosis. The unexpected high toxicity was attributed to the presence of conjugated forms of mycotoxins, possibly generated by the plant metabolism, which were undetected by commonly used analytical methods. These derivatives could be hydrolysed to the precursor toxins in the digestive tracts of the animals or could exert toxic effects comparable to those imputable to free mycotoxins. Little is known about the occurrence, bioavailability and metabolism of these compounds so that a comprehensive study is needed.
This project aims at the following objectives:
Develop reliable extraction, purification and quantification methods based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of free and masked Fusarium toxins in cereals and cereal-based foods and feeds;
Provide quantitative data concerning the occurrence of masked Fusarium toxins and the circumstances in which they are formed, mainly the glycosylated metabolites of deoxynivalenol (DON), zearalenone (ZEN) and T-2 toxin (T-2) in cereals and cereal-based foods and feeds;
Assess the extent in which animals and humans are exposed to masked mycotoxins and carry out a preliminary risk evaluation.