Final Report Summary - MYCOHUNT (Rapid Biosensor for the Detection of Mycotoxin in Wheat)
Most mycotoxins are known to contaminate hazardously crops and consequently animal feeds and food products, causing significant economic losses associated with their impact on animal and human health, animal productivity as well as domestic and international trading. The European Union is setting stricter and stricter limits of mycotoxin concentrations in grains. Deoxynivalenol (DON) is one of the mycotoxins that are formed by a number of species of Fusarium, it is often formed before harvest when crops are invaded by certain species such as F. graminearum and F. culmorum.
Mycohunt project targets the development of a new system incorporating a wheat dust sampling tech-nique guaranteeing a 90% bulk transparency to detect the infection of DON in wheat grains. This MY-COHUNT system incorporates (i) a wheat dust sampling unit and (ii) an immunosensor unit where DON infection is detected by the application of immunosensor with DON-selective antibodies. The system is equipped with a user-friendly computer based control and monitoring unit. The system was fully designed based on the specific needs of grain producers, traders and other relevant players of the sector.
The concept of testing DON from wheat dust has been proven via defining the correlation between DON found in wheat grain samples and in wheat dust samples. The results of dust showed lower level of uncertainty errors than the results obtained when measuring DON in wheat grains. DON selective antibodies have been developed to be used in the immunosensor unit and a specific assay has been developed to obtain the immunosensor characterization. The components of the prototype have been constructed and integrated into a complete system including the sampling unit, the sensor unit and their control system. After testing the system at laboratory level to validate the technical requirements, MY-COHUNT was installed at the end-user facilities where field tests were performed to demonstrate the operational and functional performance of the complete system. Based on the test results the system was fine-tuned and the procedures of the optimal performance were defined. Final results, optimization measurements including lessons learnt and recommendations for the future commercial MYCOHUNT system were reported to enable results owners to achieve good market exploitation potential.
In the future, the MYCOHUNT system can be adapted for the detection of other mycotoxins in wheat or in other crop types such as maize.
Project Context and Objectives:
Mycotoxins are naturally occurring secondary metabolites produced by certain moulds/fungi as a result of their organic processes. Unfortunately, most mycotoxins are known to hazardously contaminate crops and consequently animal feeds and animal products, causing significant economic losses associated with their impact on animal and human health, animal productivity and domestic and international trade. While the economic effects are not easily calculated due to the several participants in the grain sector, European Union is setting stricter and stricter limits of mycotoxin concentrations. Deoxynivalenol also known as DON or vomitoxin is one of about 150 related compounds known as the trichothecenes that are formed by a number of species of Fusarium and some other fungi. It is nearly always formed before harvest when crops are invaded by certain species of Fusarium such as F. graminearum and F. culmorum.
Currently, there is no on-site, automatic sampling and measuring equipment for the detection of DON in cereals, especially in wheat. Using the existing sampling standards and regulations require time and result in costly decisions, while analytical techniques are either time consuming or more sensitive to cross reactivity.
Our goal is twofold: -developing a new sampling technique guaranteeing a 95% bulk transparency, -adapting a biosensor technology for the detection of deoxynivalenol. The electrochemical detection was selected as the amperometric sensor technology using a special biorecognitive layer proved to be the most reliable, low-cost method to be used in an on-site operating device. Our proposed solution will provide an easy-to-use, environmentally friendly, continuously operating system to fight against the mycotoxin infection.
The Mycohunt project aims at increasing the competitiveness of a large group of SMEs by developing a cost-effective method to detect infection of mycotoxin deoxynivalenol in wheat grains which is a major threat to the food and feed sector of the European industry. A group of SME-AGs, covering the two relevant sectors and representing vast number of sector SMEs, have put together this project in order to gain knowledge and resources to further exploit the results of the novel technology proposed by providing a thorough sampling and measurement method of grain.
The economic consequences of mycotoxin infection to animal husbandry and the cost of infected crop to farmers as well as mill and storage house owners are of increasing concern while human health and safety effects are and must be addressed additionally.
• To develop effective, non-destructive sampling apparatus in order to assure adequately representative measurement aiming at 90% correlation with the bulk samples.
• To assess detection limit of 50 ppb, efficacy, specificity and sensitivity of the sensor
• To develop a data evaluation algorithms and to set up correlation databases
• The understanding and determination of parameters (temperature, pressure, vacuum, etc) affecting the sampling precision avoiding the damaging of grain.
• To develop immobilising methods for sensitive antibodies, and assess different approach-es to enhance sensitivity, reproducibility, life-time, detection range, detection limit, and/or other specifications.
• The investigation of the cross-reactivity of other trichothecenes such as 3-acetyl-DON, 15-acetyl-DON, nivalenol, etc.
• To realize a commercial price for the sensor while keeping the price of the whole apparatus with the sampling unit excluding installation cost.
TRAINING, DISSEMINATION AND EXPLOITATION OBJECTIVES
• The development of training activities addressed to the SME members of the SME-AGs, first in pre-validation stage in order to ensure the understanding of the technology opera-tion before the partners initiate the validation tasks.
• Technology transfer will be carried out to present the project results.
• To develop a sound dissemination plan to deliver at least 4 workshops, presenting the re-sults of the project in European conferences and specialised magazines
Introduction into MYCOHUNT
The main result of the whole project is the MYCOHUNT Technology that is a fully automated device providing efficient, truly representative immediate on-site testing of DON toxin (deoxynivalenol) in wheat enabling traders, mills or growers to monitor crop quality in their large-sized lots. The MYCOHUNT system incorporates (i) a wheat dust sampling unit and (ii) an immunosensor unit where DON infection is detected by the application of immunosensor with DON-selective antibodies. The system is equipped with a user-friendly computer based control and monitoring unit. The present description introduces the public information on the steps how the final output of the project was realized.
Work started with the system specification based on the relevant market needs. First, the corresponding European regulations and current methods were summarized; essential background information was provided for market analyses. The market need was examined from two sites. On one hand a questionnaire was prepared, which was sent to partners and translated to the languages of the consortium members. The answers were collected continuously and the first results are now summarized. The results of questionnaire survey were continuously up-dated on the confidential project web pages. On the other hand, the market needs were analyzed, based on the experience of consortium members and on available public information. The results of the survey was taken into account during the system development.
In parallel to the system specification further activities started in September 2010 to define the correlation between DON in grain and DON in air/dust. First, the laboratory scale transport aspiration facility for dust collection was set up based on previous tests and then the standardization process of the grain dust sampling method – defining particle size and amount of dust collected in fresh harvested wheat was completed. The results showed that approximately 1.5 % of the sample was smaller than 2.5 mm and this is defined as small grains; when sieved on the vibratory system, 1.5 % of this fraction was found to be smaller than 1 mm. The first field tests on wheat varieties with different spectrum of Fusarium disease resistance was started at the experimental fields of HOGENT and generated samples for the determination of correlations between contaminated wheat grains and dust. Further wheat samples were provided by SME-AG members for testing. The first eleven wheat samples had very low DON concentration in the grain particles (Belgian, German and French origin). Two Hungarian wheat samples had a high DON concentration in the grain particles (1 to 3 ppm).The results showed that DON concentrations in dust and chaff were higher than in the grain particles. Further tests were performed during the second period. The results underlined the existence of correlation. Furthermore, artificial contamination in the research plots of HOGENT was repeated to obtain wheat samples with high DON content and the correlation methodology was further tested under field conditions. To provide the essence of the immunosensor antibody development and characterization started with synthesis of immunogens and enzyme labels which was fulfilled. Ethically approved female mice were immunized with varying amounts of DON immunogen. The cell fusion process was successful, the spleen cells of the first immunized mice were fused with murine myeloma cells. As a result of these activities the first milestone of the MYCOHUNT project about obtaining DON-immunogens, DON-enzyme labels and immunised mice with good response, was successfully achieved. The work continued with further mice immunizations, cell fusions and hybridoma screenings to gain monoclonal antibodies for characterization. As a result DON selective antibodies have been selected successfully and have been purified. Their mass production continued to provide sufficient amount for the immunosensor measurements. Parallel to the characterization further potentially selective antibodies were under development by UGENT providing an added value. The design of the sampling unit of the MycoHunt system has been completed. The original idea on the dust collection and sampling method was discussed among consortium partners based on previous research findings. As the vast majority of dust particle size turned out to be more than 1mm the original design was slightly modified accordingly, since some previously planned parts turned out to be unnecessary. The optimal combination of the different design versions for each part was chosen and based on the design and associated construction plans the parts and units of the sampling system were constructed. The final version of the complete system design was provided to partners
In the workfram of immunosensor development and characterisation, first the electronic design of the deoxynivalenol amperometric sensor unit was made. The development of the automated system for the immunoassay of DON based on the concept of sequential injection analysis (SIA) with an amperometric biosensor was developed for the laboratory measurements in ULUND. The system was designed to be fully computerized and suitable for the on-line monitoring and quantification of deoxynivalenol. This system was successfully optimized using Zearalenone (ZEN) as model analytic compound, until DON antibody has become available. Interim results underlined that the fully automated flow immunoassay was achieved. Optimum system and good performance were obtained and the system was ready for DON detection. As soon as ULUND received the DON antibody from UGENT, the knowledge gained from studies on ZEN were transferred to DON under laboratory conditions. Further work was done to develop multivariative analysis strategies for the elimination of unspecific signals. Apart from obtaining good performance with the system adapted for DON detection, great emphasis was laid on the reduction of time duration of the immunoassay by all possible means.
Once the prototype parts and individual components were constructed work continued with their integration resulting in the complete MYCOHUNT system. Parallel to the system integration, the control system to operate the MYCOHUNT prototype has been successfully designed, implemented and became ready for fine-tuning. Once the sensor unit for DON detection was developed the integrated system became ready for testing and validation. The transportation and the on-site setup and installation of the prototype were finished by the end of October 2012.
In WP7 the system validation was performed at laboratory and at industrial scale. Testing of the operation of system units and parts was performed until the system became fine-tuned for performing the field tests during wheat harvesting and storing. Before the 2012 winter season end, on-site dust aspiration tests were performed to adjust the operation of the sampling unit and the control system and to validate the technical requirements. On-site work continued early spring 2013 while in the meantime subunits and different components of the equipment were tested under laboratory conditions to enhance and validate measurement accuracy and appropriate operation. The validation of the technical performance of the prototype at laboratory level has been completed. Once the flow cell for DON detection was developed it was inserted in the sensor unit. Demonstration activities then continued with validation measurements with the completely integrated prototype including the DON specific flow cell and then with performing field tests with the final aim to smoothly operate the system both under manual control and under fully automatic operation of the sampling and DON measuring processes. The work ended with the industrial validation and system fine tuning via performing field tests with the prototype during the summer harvest. Final results as well as optimization measurements including recommendations for the commercialization of the MYCOHUNT system were provided to result owner SME-Ag and SME partners.
As a supportive output a guideline on prevention measures was developed as the result of the work of HOGENT in close cooperation with SME- AG and SME partners. The guideline aims to offer very clear information on the development of mould and mycotoxins and the control measures to guarantee the quality of stored cereals and the successful monitoring of mycotoxin contamination at any level of the cereal commodity chain. Depending on the SME Association members’ needs the documents have been translated to several languages. During the final project meeting non-RTD partners decided to make Annex I. ‘Guidelines for prevention and control of mould growth and mycotoxin production in cereals’ and Annex II. - Training tool PPT available to the public as they have no confidential content. These documents can be used by non-RTD partners to develop further simple and clear dissemination tools to reach farmers and traders at a wider level.
The primary project result is a pre-competitive prototype that was crafted by the RTD partners to provide an innovative, accurate and user -friendly measurement device to the participating SME-AG and SME partners that is able to detect mycotoxin contamination in wheat by its novel biosensor, targeting the market of farmers, mills, grain traders, suppliers and distributors. Based on the final results, MYCOHUNT SME-AGs and SMEs intend to break into the mycotoxin-detection measurement market in Europe which is estimated at several hundreds of billion EUR. Thus the consortium SME-AGs and SMEs that cover the full supply chain want to take an advantage of early market penetration and outrun the existing competition. Since MYCOHUNT is a long-term investment, the partners have analysed the benefits they expect to receive from a business perspective and based on identified exploitation opportunities, a consolidated business plan was set-up following a conservative approach. The SME-AG partners (HGFA, CESFAC, ASEMAC, SEEDYZ and SYNAGRA) will benefit from the royalty fees from sales of the MYCOHUNT system and services and royalty fees from third parties. It was agreed at the final meeting that after each sold MYCOHUNT system, 1% of the sales price will go to each of the SME-AG partners. They will also gain additional indirect benefits from avoiding economic and social losses caused by mycotoxins through their SME member network. Technical SME partners OSV, EST, IMPULS and ETIA will undertake production of the new technology under a non-exclusive commercial agreement with the SME-AGs, who will receive royalties after the sales of systems and services and will help in distribution and marketing activities. All the technical SMEs (ETIA, IMPULS, EST and OSV) will undertake sales by sharing the market on a geographical based method. The main distributors of MYCOHUNT will be ETIA and OSV, having a vast experience in agricultural and sustainable technologies sales and installation, they will benefit from the manufacturing and sales of the basic components of the system and from gaining a clear competitive advantage by early access to the market before their competitors. The end-user SMEs will be able to buy one MYCOHUNT device on a reduced price of 10-40 % from the sales price. It was also agreed at the final project meeting that the MYCOHUNT prototype will stay installed in Dunaújváros, Hungary, at the premises of end-user SME DUNAGABONA, who may carry out demonstrations and initiate a free pilot service scheme for its distributers and regular customers in order to get ready for rapid and successful market entry. In order to facilitate the above mentioned rapid market entry, end-user SME DUNAGABONA suggested to create a project follow-up company, where the non-RTD partners of MYCOHUNT could be represented as shareholders for the further development and sales of the device. (for example MYCOHUNT Ltd.). One of the other important sub-result of the project is the developed antibody. The exploitation of the antibody is expected in the following way: it was agreed at the final meeting that the developed antibody can be used in other systems and every time the antibody is needed for research purposes, it is allowed by the Consortium Agreement. In addition to this, RTD partner UGENT already received commercial interests towards the developed antibody from different industrial companies, like: R-Biopharm Group (Scotland) and Bioproxima (the Netherlands). Every time there is a commercial interest from UGENT, they will ask the agreement of the non-RTD partners.
After the end of the present project the consortium deems that further development
and demonstration necessary to transform the prototype to marketable product ready to penetrate the market. Non-RTD partners estimate an additional 300,000 EUR needed for post-project development, and the working industrial unit can reach the market within 24 months of project completion. A significant slice of this investment shall be directed to branding, marketing, storing and employing the additional staff as required. In addition during further development activities, the MYCOHUNT prototype is expected to be installed in Hungary, where DUNAGABONA may carry out demonstrations and initiate a free pilot service scheme for its distributers and regular customers in order to get ready for rapid and successful market entry. The 300.000 EUR that is needed for the complete market introduction of MYCOHUNT is planned to be covered partly or fully from the Horizon 2020 programme, where special calls for proposals will be available for SMEs to market introductory activities of products and services. In case the proposers will fail to submit successful project proposal(s) to H2020, they will search for private investors, who can fund the commercialization of MYCOHUNT.
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