A new device to detect quickly and friendly Ochratoxin A and other myco-toxins in feed, food and beverage

Projektinformationen

DEMOTOX

ID Finanzhilfevereinbarung: 604752

Projekt abgeschlossen

Startdatum 1 Juli 2013

Enddatum 30 Juni 2015

Finanziert unter

• Specific Programme "Capacities": Research for the benefit of SMEs

Gesamtkosten € 2 005 718,00

EU-Beitrag € 1 295 952,00

Koordiniert durch

AUTOMATION SRL
Italy

Final Report Summary - DEMOTOX (A new device to detect quickly and friendly Ochratoxin A and other myco-toxins in feed, food and beverage.)

Executive Summary:
4.1.1. Executive summary
The DEMOTOX Project is a demonstration activity of the results obtained in the OTASENS Project OTASENS, Project No 232245), which was a Research for SMEs (R&S) framed in the FP7 European Project. The DEMOTOX Project aimed the protection of consumer health through the fabrication of innovative, low cost, portable detection systems able to monitor ochratoxin A (OTA) contamination in wine, beer and in cereal based feed in a rapid a relative way meeting the SMEs’ requirements operating in the production and trading of some food and feed commodities. In particular in DEMOTOX two pre industrial series of devices have been developed starting from two laboratory prototypes obtained in the OTASENS research project. One device, brand MyconixCHEMTM, is an industrial prototype based on a smart TLC detection system. In this system, a-Si:H photo-sensors is coupled with a TLC plate (Thin Layer Chromatographic plate) for detection of OTA fluorescence under UV illumination. The systems are able to detect also Aflatoxin B1. The other device, brand MyconixBIOTM, is an industrial prototype based on micro-immunoassay detection system where the a-Si:H photo-sensors are coupled with a surface properly treated to host an antibody-antigen reaction involving OTA or AFLA molecules. Simple protocols have been studied and set up to extract OTA from the investigated wine and feed samples, with an environmentally friendly, rapid and reliable methods. In this manner the systems of analysis above become easy to use, by allowing people in general non-expert to perform analysis even in poorly equipped places. Indeed, the pre-industrial prototypes are compact, portable, simple to assemble and do not need of chemicals, because the supplied package is completed with a kit of mixtures properly prepared for extraction of the mycotoxin (the target) and preparation of the samples for analysis. Twenty devices for each type have been manufactured and a road map has been studied to carry out demonstration tests. The performances of the pre-industrial prototypes were evaluated through functional and technical tests performed in the laboratories of the Consortium and demonstration activities carried out in in the laboratories of the Consortium as well as in external companies and in an important external laboratory (University of Milan-Institute for Food Inspection). Taking into account the technical results obtained in terms of sensitivity and reproducibility, the demonstration results in terms of feedback from the external companies and the feedback from potential market, the Consortium decides that both the technologies are valid for further industrial development. In particular it has settled that MyconixCHEM system can detect 0,2 ng of OTA in red wine samples and 0,2 ppm of OTA in cereal based feed. The MyconixBIO system is able to determine if a sample contains a concentration of OTA above or below 1ng/ml. Analysis of market and contacts with potential buyers in different sectors allow to plan a market strategy and a commercial plan, in order to exploit the results. The plan provides an improvement of the devices before launching into the market, updating of the business plan and application of direct marketing strategy. Dissemination activity has been performed not only towards stakeholders and other scientific communities, but towards a more general public, in order to inform and raise consumer awareness about the problem of the harmful effects on human and animal health by traces of mycotoxins. The development of the whole project was truly a team effort that involved the two Companies which have manufactured the pre-industrial prototypes (AUTOMATION and LUMISENSE), the two Research Centres (UNIROMA and INESC MN) for support and assistance, a company which produces fish feed (EWOS) and an organization (CREASYS) expert in market analysis and business plans.

Project Context and Objectives:
4.1.2. Project context and objectives
The concept of DEMOTOX encloses a philosophy in which prevails the cost and the ease of use of a device that is able to provide evaluation of foodstuffs in order to protect the health, in particular this concept is part of the larger concept of food and feed safety. A more safety for food and feed could be achieved through a more spread practice in the controls. Project context is to prove the viability of the results obtained as outcome of the OTASENS Project, which was funded within the program “Research for SMEs; FP7–SME–2008–1, Grant Agreement No. 232245. The results obtained in the OTASENS Project offer a potential economic advantage to the partners, because they give them possibility to develop a simple, innovative device, portable, sensitive to detect Ochratoxin A (OTA) and other mycotoxins. The SMEs leading DEMOTOX are committed to taking the laboratory prototypes obtained in OTASENS to commercial products. Thereby, the purpose of DEMOTOX is the technological transfer of research prototype devices from a research laboratory toward industrial simple products able to detect mycotoxins and by verifying their functionality according with the results obtained in the research activity of OTASENS. Mycotoxins are produced in foods, beverages and animal feed under certain conditions, often in the early stages of stowage and storage. They are toxic metabolites generated by moulds that affect the quality of feed and grain. These commodities are monitored and regulated for mycotoxin contamination. Therefore the main objective of DEMOTOX is to validate a pre-series of pre-industrial prototypes through a suitable plan of demonstration activity before to fabricate a complete commercial product. Besides this activity, dissemination activities have been performed with the double scope of inform the commodities’ producers about this innovation and public about the problem of toxin contamination along the food chain. A suitable market research has been carried out in order to test potentiality of market and competitors before to launch the products. Consequence of the training and dissemination campaign and of the market analysis is a business plane ready to be used for direct marketing and for attracting venture capitals or anyway for finding investment necessary for further industrial development.
The project has achieved most of its objectives and technical goals for the period with relatively a few deviations, as successively explained in detail. AUTOMATION and LUMISENSE have developed various versions of the prototypes before to achieve a final version of the devices. The re-engineering of the lab prototypes was not simple and it has required more time and efforts than foreseen ones. In order to understand the work carried out by the two producers of devices, it should enough to show the point of departure (the laboratory prototype, as results of the OTASENS Project) and the point of arrival (the pre-industrial prototypes) (see figures 1 and 2). For each type of system 10 devices have been produced, as pre-series of prototypes. The scope of this production was to perform a road map based on the method of round robin, by involving external end users, selected by CREASYS. With circulation of the devices among the end users, the Consortium intended to achieve two objectives: to carry out the demonstration tests at external Companies, willing to do the experiments; obtain a sort of feedback coming from the critical observations and suggestions given by the external end users.
Fig.1 The original laboratory prototype (to the left, SMART TLC detection system) and the final pre-industrial prototype (DEMO-CHEM device, brand MyconixCHEM, designed and produced by AUTOMATION).
Fig.2 The original laboratory prototype (to the left, the Micro-Immunoassay detection system) and the final pre-industrial prototype (DEMO-BIO device, brand MyconixBIO, designed and produced by LUMISENSE).
Demonstration tests were carried out besides in external companies also in the laboratories of the Consortium. The results were critically analyzed in order to improve the devices. An important part of the tests has been the procedure for extracting the mycotoxins from wine and cereals and the procedure for preparing the samples for analysis. These procedures may strongly influence the sensitivity of the instruments, thereby new methods have been developed to obtain the best results. Indeed, the end users not only require simple methods of analysis, but also simple and rapid methods for extracting mycotoxins. A detailed discussion about these performances is presented in the report.
At the moment, the industrial prototypes are not still ready for their launch into the market. However the main characteristics which were defined in the research prototypes have been maintained. Not only, but some drawbacks verified in the original devices have been overcome, by obtaining better devices, more suitable to be used by a wide audience of users, not necessarily skilled in chemistry or in analysis methodologies.
Tests for the detection of OTA in wine and grain samples have been extensively performed, demonstrating that both systems are able to determine whether a sample has OTA contamination above or below a pre-determined level. Some differences between two system can be exploited in the future: DEMO-CHEM is a device more simple and practical to be used also in field, but at the moment it can detect only the mycotoxins fluorescent. DEMO-BIO is a little more complicated to be used, also as concerns the extraction procedure, but it may detect more mycotoxins (OTA, AFLAB1 and M1, DOM, Patuline, Fumosine). Specific procedures of validation have been adopted, with differences between the two type of devices, which operate on different principles. In particular, it has been settled that the DEMO-CHEM system has a sensitivity of 0,2 ng of OTA and it is enough sampling and analyzing 10-15 ml of red wine or 50 gr of cereal based feed to establish if a sample is within the law limit in absolute OTA concentration. The immunoassay system is able to determine whether a sample contains a concentration of OTA above or below 1 ng/mL (1ppb). Moreover specific procedures of extraction from cereals have been developed in order to achieve the limit of detection of OTA well below 200 ppb in feed for some animals, as indicated by the European Directives. Practically the effort made by AUTOMATION and LUMISENSE has been to produce a prototype of miniHPLC (the DEMO-CHEM device) and a prototype of miniELISA (the DEMO-BIO device).
The two new devices allow to apply the method in premises with little or without equipment for chemical analysis. Also the dispersion of products in the environment has been greatly reduced. Ultimately these systems are compact, simple and easy to use. They are supplied with suitable chemical kits ready to perform a complete analysis. With the kits, standards of mycotoxins, according to the needs, are also supplied.
The development of the whole project was truly a team effort that involved not only the teams of the two Companies, AUTOMATION and LUMISENSE, assisted by the Research Centres (INESC MN and University of Rome “La Sapienza”), but also experience of EWOS, a producer of feed for fish and animals, and experience of CREASYS as concerns the market analysis and competitors’ analysis. CREASYS has prepared a business plan which will be the base of a suitable commercial plan.
For the SME members in the DEMOTOX Consortium there are clear and demonstrable benefits to be gained from the development of a successful technology. The expected impact of this will be to drive the SMEs within European agro-food industry and to connect them with the European policy and European regulations, in a highly competitive position in both the European and International markets. This will benefit the SMEs through product innovation and job creation and the general public through the introduction of an innovative technology to safeguard public health. The impacts will be evidenced widely, commodity producers will profit from controlling their products more diffusely without increasing too much their costs and introducing into the market higher quality products, free from toxins contamination, giving to the users and consumers animal feed and human food more safe.
In the last ten years there have been many publications and European Projects that have explored the problem of mycotoxin in feed, food and beverage. In particular, cereals, wine, grape juice and beer have recently been identified as the most important source of ochratoxin A (OTA) (see, for example, Mycotoxin Prevention Cluster). One of us, Prof. C. Fanelli from University of Rome, has presented a communication at the EXPO 2015, in Milan (Italy) on “Prevention and degradation of aflatoxin B1 in feed based on corn”, quoting the project DEMOTOX for monitoring of contamination by mycotoxins in maize through new, low cost devices, user friendly, usable also in field.
In many papers and publications some methods of analysis and monitoring of the presence of OTA in wine and cereals have been indicated, but generally these are costly, not simple to apply in all farms and not user-friendly, as already previously reported. The high toxicity and the wide diffusion of the mycotoxins, OTA, but also other mycotoxins, like Aflatoxins, Patulin, DON, have led to the definition of strict legal limits for its tolerable presence in different food commodities.
Competitiveness of SMEs which produce analysis instrumentations depends directly on introducing
technologically advanced devices into the market. The described detection systems, although in a not final commercial form, are user friendly and can be available at low cost. Socio-economic impact is direct consequence of the diffusion of such a kind of technology.
Different sectors other than the ones directly involved in the project may be interested on applying the new method resulting from the Project. Namely the sectors of the flour and meal producers for human food, the sector of dried fruits and coffee traders, and consequently the sectors of bread production and coffee roasting. In these cases it can be required to modify the sample preparation protocol according to regulations indicated on Commission Regulation (EC) No 401/2006 of 23 February 2006, laying down the methods of sampling and analysis for official control of mycotoxins in foodstuffs.
The obtained final results and their potential impact and use including the socio-economic impact
and the wider societal implications of the project can be summarized as follows:
• Closer business relationships between industry and Academia. SME participants have interacted and will interact closely with R&D organisations.
• Increased European knowledge and skills at the frontier of smart component and smart systems integration. The new systems are included in the category of smart micro-systems and will contribute to the competitiveness of the European industry involved.
• The DEMOTOX project contributes to environment protection through the improvement of crops and food. These actions can lead to a reduction of waste and, ultimately, helping to improve the health, end up providing intelligent solutions in energy management.
An important activity of dissemination has been carried out by the partners; in particular by AUTOMATION, who has presented the project in two trade fairs in Milan, EWOS, who has done
advertising campaign to its customers, CREASYS, which has contacted Associations and single producers, the Research Centres which have shown the scientific part of the DEMOTOX project in different posters and communications in congresses and conferences(see Section A), with the obvious limits of confidentiality regarding the details.

Project Results:

4.1.3. Main S&T results
The primary objective of this project is to demonstrate the viability of 2 toxin detection systems, developed within the OTASENS project, to produce a “product-like” functional prototype and to market the resulting product. Indeed, the DEMOTOX is an industrial evolution of the OTASENS Project, which started December 1, 2009 and ended on November 30, 2011. The DEMOTOX Project is a demonstration activity of the results obtained in the OTASENS Project (OTASENS, Project No 232245), which was a Research for SMEs (R&S) framed in the FP7 European Project. From the beginning of the DEMOTOX project, the project partners which have the responsibility of technological transfer a laboratory prototype into an industrial prototype has made a great effort for developing the DEMOTOX systems and fulfil the project requirements, i.e. performances of the devices. The performances have been subdivided in two groups, one qualitative and another quantitative, as listed below:
Qualitative performances
• Portability
• User friendly
• Measurement repeatability
• Reliability of the systems
• Time of analysis (including the time for sample extraction and preparation)
• Chemicals. Environmentally sustainable. No toxic waste. Extraction solutions based on water
Quantitative performances
• OTA in animal feed and fish feed. The limit of detection should correspond to the directives of the EC and/or to the Directive of Italian Ministry
• Limit of detection for AFLA in feed. As indicated by the EC directives
• Commercial device: low cost and short time to market
• Certification validation
As conclusion of the activity of Project, two pre-series of industrial prototypes were manufactured and tested, made with different technology: a pre-series of DEMO-BIO devices, made by LUMISENSE, based on industrial micro-immunoassay detection system, like a mini-ELISA. Its brand is MyconixBIO. A pre series of DEMO-CHEM devices, made by AUTOMATION, based on liquid chromatography, like a mini-HPLC. Its brand is MyconixCHEM.
New procedures to extract OTA and other mycotoxins has been proposed by the teams of the two Research Centres which have assisted AUTO and LUM in preparation and assembling of the devices. One of these methods is in the process of patenting. Registration of patents related to the devices or some their components are in progress.
At this stage, all project delays have been properly addressed by the project partners by applying suitable actions to mitigate the risks indicated in ANNEX I and without affecting the overall project schedule, but above all the main result. All deliverables have been officially and successfully reported (submitted). All technical tasks have been also successfully executed and all project milestones achieved.
WP1 (1st -6th month), Demonstrator manufacturing technology.
All WP1 deliverables have been successfully submitted: D1.1 D1.2. Milestone MS1 has been verified in the provided time.
20 devices have been manufactured as demonstrator devices: 10 of these – named DEMO-CHEM - are based on the prototype developed during the OTASENS Project, by the RTD Performer UNIROMA; other 10 – named DEMO-BIO – are based on the prototype developed by the RTD Performer INESC-MN (here referred as INESC MN). In particular DEMO – CHEM is a thin film chromatographic –based system. DEMO – BIO is a micro-immunoassay- based system. WP1 has been completed in the first period, as planned and the objective achieved within 6 months. During assembling of the devices, some parts and components (for instance sensors, microfluidic chips, electronics, software, design of the container box, filters, etc.) have required an accurate selection and some separated tests in order to achieve a first version of the devices. The external container has been manufactured by 3D printers.
WP2 (3rd -22nd month), Robin round demonstration tests
All WP2 deliverables D2.3 and D2.4 have been successfully submitted, although not in the provided time. Indeed, D2.3 - Mid- term results of round robin procedure- has been submitted on 03-10-2014 instead of 30.06.2014. D2.4- Collection of results- has been submitted on 15 June 2015 instead of 30 April 2015.
The WP2 has been extended until the end of Project (24th month). Therefore we can take into account the job by increasing the number of person months (organization of the tests, laboratory tests, setting of the devices, assessment, adjustment of the devices). For instance, to carry out the demonstration activity AUTOMATION has employed more person months with respect to the provided. INESCMN has employed other people and more person months. Neither has charged costs greater than provided ones.
The objective of this workpackage is to identify the strategy, design the experimental procedure to be implemented, organize the methodology for the collection and analysis of the results and perform the systematic tests of the demonstrators developed in WP1. From the point of view of the validation of the systems, this is the most important workpackage.
In the first period the following steps have been performed:
1st STEP: demonstration tests conducted by AUTO and LUM in their own laboratories, in collaboration with UNIROMA and INESCMN respectively.
2nd STEP: verification of the performances of both the demonstrators in the same laboratory, for comparison purpose. This verification was performed in the laboratories of INESC MN in Lisbon, on February 26th , 2014.
3rd STEP: reduction of defects, drawbacks, improvement of functionality and initial optimization of devices. This step has determined the delay of the deliverable D2.3.
At the 9th month the following deviations have been registered:
Task 2.1 no timing deviation.
Task 2.2 no timing deviation, but deviation in terms of activity. Indeed, in the first months of this activity, inside the activity called “Experimental test execution”, the tests of laboratory, carried out in the own countries and in Lisbon have been included. Delay in sending the devices to EWOS and to the end users has produced a consequent deviation in the subsequent tasks.
End users outside the Consortium has been selected in the first months of the Project. The partners have defined the procedure of the round – robin method, which should be used to perform the tests and verify performances of the devices. In the next months of the first period (from 6th to 9th month) the main activity has been to carry out experimental tests in the laboratories of AUTOMATION and LUMISENSE with the devices fabricated by each Company. Only standards have been used. At the end of February the technicians met in Lisbon, where both the devices have been tested also by using samples contaminated by OTA prepared and supplied by EWOS. These tests were necessary also to discuss and correct some drawbacks previously found during preliminary tests. The session was employed also to do a short training for AUTOMATION, LUMISENSE and EWOS, performed by UNIROMA and INESCMN.
After the selection of the external end users, in the second period of Project (01/04/2014-30/06/2015) the procedure of the round – robin method originally defined among the partners has been subdivided in two phases. This, in consequence of the long job of experimental tests carried out in the internal laboratories of the Consortium. On the basis of the results of these tests, the producers have gradually modified the original version of their devices.
The first phase of the round robin procedure has been performed at the external producers (sectors of the wine and cereals) with the assistance of the Consortium. This phase is based on the qualitative validation of the structural characteristics of the devices: portability, ease to assemble and use, method of analysis, compactness of the system. The second phase was thought in a first moment as a procedure of demonstration without assistance, but in any case by involving the external end users. However, this idea has been dropped for strategic evaluation, in order to mitigate the delays. Thereby, the second phase of the round robin procedure has been modified by introducing a intense activity of experimental tests carried out in the lab, with the scope of a further optimization of the devices. Practically at least 5 different versions have been produced for each type of device in order to overcome flaws and drawbacks encountered gradually. Therefore, the second phase was based on the validation procedures suitable to validate the instrument and validate a specific screening method. The results obtained with these systems on samples prepared both with the procedure developed by UNIROMA and with the procedure standard used by EWOS have been compared with those obtained by using traditional analytical methods, by finding a good correspondence. Also an external Institute of validation of secure skills and experience has been involved (University of Milan). Some samples has been extracted by using a new method of extraction developed by INESC MN. Operating Manuals, already included in the Final Report of OTASENS Project, have been updated, also taking into account of the results obtained “ on field” with the demonstrators, in the first phase of the round robin process. The procedures of extraction described in previous manuals have been also updated. Both the devices have been tested to verify if they can detect other mycotoxins, in particular Aflatoxin B1. Indeed, both the devices are able to detect this mycotoxin, without particular modifications to the systems.
By summarizing:
a) Delivery to EWOS of some demonstrators for laboratory tests and comparison with traditional techniques. EWOS has analyzed several samples with an HPLC-MS/MS based multi-myctoxin method, positively identifying fungal metabolites and the respective concentrations. The same samples have been used for carrying out analysis with DEMO-CHEM and DEMO-BIO in July 2014.
b) 1st phase of the round robin activity performed at the external end users, selected in Italy (wine and cereal producers) and in Portugal (wine). Mid-term results are reported in the Deliverable D2.3. Comment and suggestions are also reported and are evaluated positively.
c) 2nd phase of the round robin activity performed at the laboratories of EWOS, AUTO, LUM, UNIROMA and at the Department of Veterinary Science and Public Health, University of Milan-Faculty of Veterinary Medicine, Institute for Food Inspection, Lab. Food Safety.
d) With DEMO-CHEM it is possible to detect aflatoxins (type B1, AFB1) without significant modification to the apparatus, because the frequency of emission of light from excited AFLA is almost the same of OTA. In INESC MN, the extraction procedure has been tested and proven to work for both OTA and AFB1. The extraction of AFB1 from fish feed and detection with DEMO-BIO did not give any problem and it is confirmed that AFB1 can be detected with DEMO-BIO in spiked buffer solution (with similar results as OTA). Of course, the concentration of the extracted sample and sensitivity of the instrument must be increased, because the limits of detection for AFLA B1 defined by the Directives for wheat, maize and other cereals are very low, less than the limit of detection indicated for OTA. As concerns other mycotoxins, for instance DOM and Patuline, DEMO-BIO shows an higher versatility. However for detecting these mycotoxins, some attention has to be paid.
WP3 (12th – 24th month). Result analysis
All WP3 deliverables D3.5 and D3.6 have been successfully submitted, although not in the provided time. Indeed, D3.5 – Critical analysis of demo tests- has been submitted on 02-03-2015 instead of 31.12.2014. D3.6 - Final results of demo tests- has been submitted on 09 July 2015 instead of 30 April 2015. MS2 (on 14th month instead of 12th month): the Consortium decides to pursue the project, in spite of some drawbacks, but overall with important results. MS3 (on 22nd month instead of 20th month): the partners have verified the results of analysis of comparison. Although the results have been collected with delay, however they confirm reliability of the pre-series of prototypes, although with some flaw (reproducibility and sensitivity). Software has been validated by the users (functionality of the devices, operating manuals, etc.) and in the lab.
The results of the demonstration tests are strongly connected with the procedure of analysis provided in WP2 and with the analysis carried out: the demonstrators DEMO-CHEM and DEMO-BIO are tested in the laboratory by AUTO and LUM respectively, which act in synergy between them exchanging their manufactured devices and discussing about modes of performing the tests. Various versions of each device have been developed during this work package (Task 3.1) in order to achieve simple and reliable systems. Initially, in the case of DEMO-CHEM, traditional eluent was not compatible with the material of the small pumps used, therefore it was necessary to change the eluent and calibrate again the device. In the case of DEMO-BIO some drawbacks have been found in the part of the microfluidic channels and in the procedure of injection of the solutions. EWOS has performed the first controlled field tests and has compared the results with those obtained in the laboratory by standard techniques (Task 3.1). Further corrective actions have been taken (Task 3.2). Analysis has been critically performed during the collection of the results (Task 3.3). This activity has been made gradually in order to verify the errors, prepare other chemical kits and improve the method of data collection. After completion of the tests according to the “round-robin” method, risk analysis have been estimated in view of the development of industrial products (Task 3.4). Suggestions by end users have been taken into account and possible corrective actions have been taken (feedback, Task 3.5). As final comment about the results of this WP, the Consortium can say to be very satisfied of the job performed, in spite of some drawbacks which however are not sufficient to invalidate the project and do not impinge its critical objectives.
The steps performed can be summarized as follows:
1st Step. Standard techniques (HPLC-MS / MS) applied to verify the real concentration of OTA in the extracted samples. An extraction process is always necessary in order to obtain samples containing as few interfering compounds as possible. However this procedure must be evaluated in terms of efficiency, depending on the mixtures used. The extractive methods developed during the DEMOTOX project are a little different from traditional ones. The goal achieved has been to obtain methods simple, fast, reliable, easy to use and, above all, devoid of unsafe solvents. Practically they are environmentally sustainable. It should be noted that in the package provided to potential buyers, in addition to the apparatus of measurement is provided a chemical kit specially prepared to perform the extraction and preparation for analysis of the samples with specific substances suitable for the measuring systems.
2nd step. Data collection of the tests carried out at the following external Organizations:
- AIRES, (Associazione Italiana Essiccatori Raccoglitori Stoccatori di Cereali e Semi oleosi), that is an association of Italian dryers, pickers and pallet stackers of cereals and oilseeds.
- Adega Cooperativa da Região de Monção (www.adegademoncao.pt) that is one of the main producers of Vinho Verde in the Minho region of Portugal.
- Consortium ASTI that collects all Italian producers of wine "Asti Spumante DOCG".
- UNIMI, Department of Veterinary Science and Public Health, University of Milan-Faculty of Veterinary Medicine, Institute for Food Inspection, Lab. Food Safety.
Specific questionnaires have been prepared in order to collect the comments and suggestion of the above organizations. In table 1 and Table 2 are summarized the answers.These are all positive.
DEMO-CHEM. Tests carried out by AUTOMATION with asistance of UNIROMA
AUTO has developed gradually several versions, made for overcoming some defects and drawbacks of each previous version.
The runs have been firstly made with standards OTA in methanol at the following 4 concentrations:
0,1 ng; 1,0 ng; 2,5 ng; 5,0 ng.
The mobile phase in this case is more simple than in the case of samples extracted. Indeed, the solution used in all the cases has been a mixture of ethanol, water, acetic acid, 4:6:1 (v/v/v)
For each concentration, 3 runs are carried out, each with 5 different devices. One of these devices (N.o 1106) has been used in the laboratory of University of Milan. A commercial software of elaboration of numerical data (Calisto) has been used to obtain the graphic forms of the responses. Reproducibility of the measures has been verified and it may be evaluated to be sufficient (see below).

DEMO-BIO. Tests carried out by LUMISENSE with assistance of INESC MN
LUMISENSE has developed the prototype with a new chip design, and a package that includes the photodiode, pumps, fluidic connections, and the control hardware and software.
Main hurdles have been overcome:
- the microfluidic acrylic chips has a different protein adsorption behavior than PDMS used in the lab prototype. The macro-machined dimensions put the assay in a different regime than with the microfluidic chips;
- a new pump has been designed to minimize flow fluctuations that generated large noise in the output signal.
- Mycotoxin extraction and purification is achieved using a process developed by INESC MN and takes approximately 15 minutes. Extraction of corn product contaminated with OTA at 44 and 17ppb give samples detected as positive by the DEMO-BIO instrument. The instrument time of analysis is now approximately 15 minutes for a total time of 30 minutes for extraction and analysis. LOD (Limit od Detection) can be lower than 17ppb for naturally contaminated samples.
- OTA extraction and matrix cleaning is at this moment done outside of the chip. A simple aqueous two phase separation process can be used, which is fast and allows for OTA immune-quantification in complex samples. Validation of the demonstrators and method of analysis have been performed on the basis of a screening method according of specification given by COMMISSION REGULATION (EU) No 519/2014 of 16 May 2014 amending Regulation (EC) No 401/2006: “Methods of sampling of large lots, spices and food supplements, performance criteria for T-2, HT-2 toxin and citrinin and screening methods of analysis (Text with EEA relevance)”.
The number of samples for validation consist of:
20 homogeneous negative control samples;
20 homogeneous positive control samples that contain Ochratoxin A at the STC (screening target concentration, selected by the operator).
They are spread over 5 different days and with 5 different users.
In order to realize a first assessment of the DEMO-BIO equipment, concerning its applicability to detect Ochratoxin A, a preliminary study was performed in aqueous buffer solution (PBS 7.2) with and without 50 ng/ml of toxin.
In this case the 20 blank solutions give an average value of 93 with only an RSD of 3% attesting the low variance obtained between assays. The same assays performed in the presence of 50 ng/ml output an average value of 32 and an RSD value of 11%.
When using 20 samples in total, only one sample (5%) is allowed to be false compliant according to the guidelines for screening methods. This condition is obeyed for the results obtained in buffer solutions.
The same approach used for validation in buffer was used for the validation with cereal feeds. Cereal feed spiked with Ochratoxin A (1) and natural contaminated feed (2) have been tested.
In the first case (1), the 20 blank solutions give an average value of 80 with an RSD of 9%, showing the matrix effect in contrast to results obtain with the buffer solution. The same assays performed in the presence of 50 ng/ml of spiked feed cereal reveal an average value of 44 and an RSD value of 6%.
In the 2nd case (2) with natural contaminated cereal feed, the 20 blank solutions give an average value of 79 with only an RSD of 7% while the samples contaminated with 44 ppb give an average value of 24 and an RSD value of 50%.
The rate of false positive is calculated according to the regulation for the different tested commodities. The rate of false positive is almost inexistent, near zero, attesting the fitness of purpose of the method.
Details are reported in the deliverables D2.4 D3.6 and in the Annex 10 (MyconixBio Overview).

In conclusion, the last results and generally the experimental work performed for evaluating the performance characteristics of DEMO-CHEM and DEMO-BIO can be summarized as follows.

The DEMO-CHEM device (brand MyconixCHEM)
A long and accurate experimental work has been performed for evaluating the performance characteristics of a DEMO-CHEM device. Qualitative method has been applied to validate the reproducibility of the measurements on a single device and on several devices. The devices demonstrate a good reproducibility as concerns the chromatographic runs. The demonstration tests have been carried out with standards of OTA at 4 concentrations: 0,1 ng; 1 ng; 2,5 ng; 5 ng.
10 devices has been produced. 7 devices has been tested in different laboratories as concerns their viability and reproducibility.
With the same device, reproducibility of the tests is around 90% ± 1, independently from the operator.
With different devices reproducibility is 50%±5, i.e of 5 devices only 2-3 give the same results. The analysis of the causes of this variability confirms that for the most part it is attributed to variability of the characteristics of the sensors and for other part it is due to variability of the TLC plates for the same mobile phase (for instance poor homogeneity of coating of silica gel on the plates). It is well known that TLC is generally used for qualitative analysis of reaction products and the external temperature could influence the rate of the mobile phase.

The DEMO-BIO device (brand MyconixBIO)
Screening assay was capable of detecting Ochratoxin A at the maximum level set for feed cereal using barley as sample matrix.
During the validation procedure it was noticed that different demonstrators have different sensibility.
The results point to that the DEMO-BIO device allows to do assays as a screening method for regulated mycotoxins, prior to send the samples for confirmatory instrumental analysis (like LC-MS/MS). This method can enhance throughput by avoiding the unnecessary submission of actual blank samples to the confirmatory method. Furthermore the transportable instrument may perform screenings in a simple equipped on-site or mobile laboratory. This way, raw materials for food and feed can already be analysed prior entering the food and feed supply chain.
The rate of false positives is very low. This result confirms general applicability of the test to separate samples with ochratoxin A at the target levels (EU maximum permitted levels) from blank samples. In conclusions the results obtained for the DEMO-BIO validation confirms the fit-for-purpose, as a screening method for OTA detection in cereals, with detection levels of 50 ppb well below the legal limit of 250 ppb for feed cereals.
WP4: (1st – 22nd month) Market strategy
All WP4 deliverables D4.7 D4.8 D4.9 and D4.10 have been successfully submitted, although not in the provided time, except D4.7 – Critical analysis of demo tests- and D4.8 - Selection of other end users- . D4.9 - Preliminary results of market research- has been submitted on 18-11-2014 instead of 31.08.2014. D4.10 - Final business plan- has been submitted on 05 June 2015 instead of 31 January 2015.
MS4. Final Check on Results. Deviation. Shifted to 22nd month. In the conference calls held in April 2015 and May 2015 and in the final meeting held in June, hearing the opinions of AUTOMATION and LUMISENSE, verified the results of demonstration tests of the first phase at the end users and the results of the second phase at the laboratories, taking into account the market research and feedback of the respondents, Consortium decides that both the technologies are valid for further industrial development.
MS5. Evaluation of dissemination and feedback actions. Final survey completed and data quality validated. No deviation. On 22nd month, at the same conference calls of April 2015, taking into account of the results of dissemination, contacts with many producers, press communications and dissemination activity (see www.demotox.eu or www.demotox.it page of dissemination), the Consortium partners evaluate quality of data suitable for a next advertising campaign, ahead of the launch of the product. The Consortium believes it is appropriate continue to act both at the level of communication and scientifically even after the end of the project pending final products on which AUTOMATION and LUMISENSE intend to carry out further additions and improvements. The efforts made so far will be fully exploited in view of the market.
In order to obtain an accurate evaluation of the market, in the first period analysis of the competitors has been performed (Task 4.1) on the basis of a flexible concept of competition, identified at different levels according to the similarity of the instruments and products compared to DEMOTOX devices.
From this analysis some main advantages of the DEMO-CHEM and DEMO-BIO demonstrators appear clear, with respect the competitors:
- Portability,
- miniaturization of the devices,
- the devices can be used really by not expert people so they can be applied outside of a laboratory.
The real competitors of the DEMOTOX devices are mainly those systems which can compete in terms of the performances above. These, in the SWOT analysis, are real advantages, because they are BCB (Basic Consumer Benefits).
In the first period various commercial strategies have been taken into account, in order to prepare a credible commercial plan (Task 4.2). These arguments have been treated in the deliverable D4.10 (Business plan) and D6.16. They are detailed in the Annex “Core of 2nd Periodic Report” . A “demo” and a business case are activities performed in the second period (Task 4.3) as basis for a credible commercial plan.
The "DEMO" is the set of information that have been communicated to the respondents through a specific questionnaire to describe the DEMOTOX devices (see deliverable D4.10 and Annex 8) and to formulate questions to understand the approval of the devices. The DEMO has been built in close relation with the sequence of the questions in the questionnaire and then it has been prepared on the basis of information already available or gained from partners. Based on concept cards reading (see Annex 8), the respondents give a certain level of agreement for both the devices. The agreement level (a.l) is calculated as difference between high agreement responses (very much and quite) and low agreement responses (A little, Not at all), where “Quite” and “A little” are counted at half. A.L. ranges between -100 (%) and +100 (%). On the whole, then, the respondents have confirmed the key properties of the devices.
To prepare a “demo” and a “business case”, first of all it has been necessary identify the type of potential customers. On the basis of the questionnaires sent to the respondents, four classes of customers have been identified:
Class 1.Hi-tech no external labs. They have instruments to perform high precise detection testing (HPLC and similar) and don’t need to send their samples to external laboratories.
Class 2.Hi tech, external labs. They have instruments to perform high precise detection testing but also use external laboratories for the purpose of certification/confirming.
Class3.Mid-tech. They are able to perform tests using Elisa methods or other similar methodology available by little investment.
Class4.Rapid tests (only). They perform only rapid test with screening purpose. Sometimes they send their samples to external laboratories for further testing.
Annual average expenditures for detecting mycotoxins have been subdivided for testing class. Class1: € 29375. Class 2: € 127857. Class 3: € 6575. Class 4: € 5890.
According to our estimates, less than 20% of total expenditure is due to internal testing, for an amount of €70 million (by 2018). An evaluation has been estimated for sector as concern internal testing only. This could be the European size of the main market of DEMOTOX devices. The internal market can be subdivide also between the Italian market, about €9m, and the Portuguese market, with about €1m. The classes of the potential customers can be considered market segments.
Besides the on-field use, it can be assumed that the devices developed in DEMOTOX could have a role even in the activities of specialized laboratories or for specific functions (for instance, MyconixCHEM in-lab screening test, tests for low-spending customers, use for teaching activity).

The “DEMO” describes a innovative device as well as it will be at the moment of its launch on the market, so if some functionality are not yet developed but they will surely be implemented before launch, they have been included in the description. The key topics of discussion have been:
A. Space needed by the new devices, accessories on the work surface, ease to use.
B. Sample extraction procedure: the requirements for a more simple and solvent-free extraction have been drawn.
C. Duration of the analysis. The consortium has become aware of the fact that DEMO-CHEM takes less than 5 minutes of time-operator to do analysis, while the goal of a total duration of analysis with DEMO-BIO is set at 15'
The “DEMO” consists of five concept cards embedded in the questionnaire (see Annex 8, the Concept Cards). Based on concept cards reading we can classify the responses and define the elements for a business case.
The new devices have been tested with external end users interested to apply more simple systems and cheaper, but at the same time by maintaining sensitivity, reproducibility and low time of analysis. Another condition has been satisfied and it is a greater simplicity in the procedures to extract mycotxins from the product, both solid and liquid.
The first tests have been carried with standard samples to demonstrate that the devices are portable, user friendly, compact, and allow short time to extract and do the analyse (advantages, strength in SWOT analysis). Other conditions are not completely satisfied: with these devices it is not possible to detect more than one mycotoxin and perform more than a single analysis(disadvantages, weakness in SWOT analysis). But the Companies involved to develop these systems are designing improvement or solutions which could solve these drawbacks. In the Annex 8 the main concepts regarding the devices and their performances have reported. The cards of these concepts are also explained as follows.

Concept A (the DEMO-CHEM device)
Description of the characteristics of the device and its use. As reported in deliverable D6.16(PU) this device may be considered similar to a miniHPCL, because it has a part that imitates the chromatographic columns ( the plates TLC – Thin Layer Chromatographic System), a part similar to a fluorimeter (UV illuminator) and fluorescence detector based on a Si:a sensors, all integrated in a system to analyze the chromatographic diagrams and to give an immediate response. The systems, in their final version for commercialization, will be completed with an automatic system to extract the myctoxin from the product which is analyzed and prepare the extracted samples for analysis. In this manner the operator may do a few preliminary operations and then the apparatus can do the procedure by itself. Other important characteristics of this device is that it does not require complex preparations and it does not use chemicals with can be dangerous (advantages, strength in SWOT analysis). The chemical mixtures are mainly based on aqueous solutions and do not produce environmental pollution. All these characteristic influence the overall cost of the device, which will be cheap (advantages, strength in SWOT analysis).

Concept B (the DEMO-BIO device)
Description of the characteristics of the device based on immunoassay methods and its use. As reported in the DEL 6.16 this device may be considered similar to a miniELISA.
A comparison is made with a commercial product. It is important to underlie that you can perform the analysis anywhere (advantages, strength in SWOT analysis) while the competitors can use their instrument only in the laboratory. Moreover the innovative device is portable and very simple to be used with respect to traditional and competitive commercial systems (advantages, strength in SWOT analysis). The chemical kit sold together with the device does not impact on the environment. All these characteristic influence the overall cost of the device, which will result lower (advantages, strength in SWOT analysis).

Taking into account the previous considerations, the business case can be presented (see Core of 2nd Periodic Report). As target of the business case it has been selected a consortium of 175 Italian wine producers. This choice is linked to the main objective of the project DEMOTOX, which is to monitor the mycotoxin OTA, mainly present in red wines. As a result of this simulation it is obtained a credible profit before taxes of €185,000 on the first year of launch and €170,500 on the second (no one device is sold at the second year).
Hypothesis for a commercial plan
Definition of a commercial plan requires to evaluate the market potentiality (Task 4.4). This is based on:
▪ Qualitative analysis of the targeted clients’ needs and current practices through the realization of asynchronous discussion groups with KOL (Key Opinion Leader) according to Bulletin Board methodology. During the Bulletin Board has been evaluated also the degree of acceptance of participants with respect to new tools DEMO-CHEM and DEMO-BIO.
▪ Quantitative survey. More than 150 associations of producers were contacted to spread the questionnaire among their members. Finally 139 completed interviews have been collected in several countries. The research, based on a structured questionnaire submitted via web (mainly) or CATI methodology (Computer Assisted Telephoning Interviews, for web non-respondents only) has been focused on: knowledge and level of awareness on the problem of mycotoxins and consumer protection, the procedures currently used to manage the risks of contamination and the related costs, price sensitivity, the degree of acceptance of new products ( DEMO-CHEM and DEMO-BIO); propensity to purchase them, acquiring any suggestions
▪ Data analysis and reporting.

The most important sectors for market research remain the cereal sector (especially maize), feed, wine and beer, but also come into play soy, pistachios, almonds, hazelnuts, coffee. According to the latest statistical data (from Eurostat, FEFAC, COPA, Brewers of Europe, OIV), the sample was therefore defined in terms of quotas and in terms of countries for each sector (table 3).
The contacts with potential buyers and the interest raised about the innovation is the basis for a business plan to be submitted to future investors. Data collection are used for direct marketing in perspective of launch into the market of the new products. Market strategy and commercial plan will be further examined in terms of costs at the moment of the launch.
The market research is based on three preliminary conditions:
1. Analysis of customer needs
2. Analysis of the competitors
3. Contacts with the producers
The preliminary contacts with the producers have allowed to complete the list of the Basic Consumer Benefits (BCB), D4.10. Further tests will allow for optimizing the systems in order to become more competitive on the market. Of course this can also depend on ability of understand the “Voice of the Customer”.
The WP is completed with the presentation of a business plan (Task 4.5) which is also reported in the deliverables D4.10 (PU) and D6.16 (PU).
WP5: (3rd – 24th month) Training and dissemination
All WP5 deliverables D5.11 D5.12 D5.13 have been successfully submitted. D5.11 Project website online and D5.13 - Final results of the dissemination – have been submitted on time. D5.12 - Mid- term results of dissemination – has been submitted with a delay of 3 months.
Only the task 5.2 “Dissemination for end users and the general public” has been extended until the end of Project. These activity will be continued also after the end of Project, for preparing the launch of the new systems into the market.
The results of this period of activity can be summarized as follows:
✓ Web site updating (Task 5.1). The website (www.demotox.eu or www.demotox.it) is continuously updated. The site has been divided in two areas, one public and one private. In the public area, the dissemination page reports any event or documents that have a bearing on the issue of mycotoxins(see deliverable 5.12 Annex4). For instance, a list of documents related to the effects of the mycotoxins, diffusion, methods of analysis.
Exploitation of the contacts with the end users and other respondents (Task 5.2). All possible stakeholders have been involved: operators in financial and commercial sectors, Consumer Associations or Groups, Industrialist Associations, SME Associations or Groups. Last but not least, dissemination has as objective to sensitize the public to the problem of mycotoxins contamination in feed, food and beverage. To this purpose, a public workshop has been organized at Rome on March 2015. The main topics used for dissemination on public information are based on the issues indicated in many occasions and deliverables. These issues constitute also a heritage of knowledge for the Consortium.
- Mycotoxin effects on human and livestock health
- Impact and damage due to mycotoxins on the crops.
- Legislation applied for mycotoxin controls.
- Methods used at the moment for controlling of the mycotoxin contamination of the feed, food and beverage
- Laboratory methods, systems on field
- Comparison of these methods and systems with the systems developed in the DEMOTOX Project
✓ Documents prepared both for presentation at fairs and for presentation to the end users. All the partners have contributed to this task.
The main performances of the new devices have been put well in light and brought to the attention in particular of producers of corn and wine through press communication, contacts with potential end users or other Laboratories and Research Centres. Public deliverables D4.7 and D4.8 describe the contacts between the Consortium and the producers of feed, food and beverage. Tools used for dissemination have been listed. Special services have been adopted for develop a big campaign of dissemination and information. For instance, a Demotox Lab Service could be designed; it could consist in a geographic distribution of Demotox analysis centers, which may serve not only small producers, but also could operate as information centers toward retails or other consumers. These D.Labs could be managed by associations or other groups of producers on the basis of franchise agreements. Each D.Lab could contact directly the Demotox technicians, as needed, through a proper multimedia Help Desk service.
As concerns development of marketing, promotional on-field campaigns, called “Demotox In Tour”, could be activated. During the DEMOTOX in Tour, technicians of AUTOMATION and LUMISENSE will travel in a camper in the various harvest sites, in order to promote the new devices of analysis for detecting mycotoxins. Each campaign will be preceded by contacts with local authorities, associations and farmers situated in the visited areas, both to publicize the initiative and to make arrangements and appointments.
✓ Specific brochures describing operating principles and instruction have been prepared (see leaflets in www. demotox.it and in Annex 11). Leaflets are also attached to the deliverable D616 (PU) and have been distributed in every occasion of events.
✓ Questionnaires for market research and market analysis

Indeed, it should be noted that the activities of DEMONSTRATION, DISSEMINATION AND MARKETING are closely intertwined and interdependent, though the dissemination activities could involve a wider range of audience, both private and public, with respect to the demonstration and marketing actions. The strategy of dissemination has been to prepare documents (leaflets, for instance) and establish preliminary contacts by starting from the direct interested (end users), then to involve the scientific community and the general public, finally to involve the authorities. Challenge is to develop systems which with a few actions and in a few time can give to the users information about the level of contamination. Finally, to general public, including the large-scale distribution (Ipercoop, Supermarkets), it is diffused information on mycotoxins and the significance of mycotoxin analysis in order to raise their awareness on the issue.
Contacts have been taken with Associations of Producers and some Centres of Aggregation (for instance in Italy the so called “Consorzio Agrario”) with the objective of disseminating of potential information to the groups indicated in the table. The intent is to contribute to make some amendments/revisions/change in the current legislative framework, that might be needed to accredit the technologies. It is reiterated here that the actions of demonstration and dissemination are interconnected between them: the objective is not only commercial (to acquire clients), but also of divulgation of new methods more practical and more simple to be used) and finally to raise the awareness of consumers on the problems of mycotoxin contamination in the food chain.
After the first phase of contacts with the end users that have carried out the assisted tests, another phase was launched to take new contacts, as listed in the table 4
The relationship with the selected associations has been treated with care for dissemination purposes during the Project and for marketing purposes after the end of the project.
As regards the public dissemination activity, both devices have been presented and announced in many occasions and events, as reported in the not reserved “Dissemination” page of the website.
It has been decided to develop a PRESS OFFICE with the scope to release press communications and to take contact with small local media to publicize the campaign; the goal is that of establishing and organizing the information on the innovation of DEMOTOX, which will be transferred to various media (Facebook, You-Tube, Twitter, etc.). Before launching the Press Office, some partners have identified, for each item, the main elements and guidelines to optimize the strategy for a correct public information. (see details in public deliverable D5.12 and D5.13). In the Annex 10 it is reported the organization chart of the Press Office process. In the same Annex a list of public event where one representative of the Consortium was present or will be present (events after the end of the Project).
As previously reported, many contacts have been established with local associations of producers, some of whom will act as demonstrators, but other subjects will act as actors who spread the information. In this contest, the recommendations of the Commission about the 1st Report of Project (Assessment Report, Ref. Ares(2014)2222178 - 04/07/2014) has been accepted (point 5f of the Assessment Report: “Use and Dissemination of Foreground. Are potential users and other stakeholders(outside the consortium) suitable involved? ")
Information has been disseminated to Policy makers, Scientific Community, the general public, specific group of end users.
Policy makers:
The performance of both of the systems (sensitivity, accuracy, precision, etc.) will be shared with the authorities. Some amendments/revisions/change in the current legislative framework might be needed to accredit the technologies.
The scientific community:
The performance of both of the systems (sensitivity, accuracy, precision, etc.) will also be shared with the scientific community. The scientific partners (UNIROMA and INESC MN) have contributed to the dissemination through scientific communications, directly or indirectly related to the theme of DEMOTOX.
The general public: Information on mycotoxins and the significance of mycotoxin analysis has been shared with general public in order to raise their awareness on the issue. (Press communications, articles on newspapers, workshops)
A specific group of end users:
The performance of both of the systems as a benchmarking report with the existing technologies will be shared with the end-users specified in D4.8.

Workshop held in Rome on 20 March 2015 was dedicated to Italian public and has obtained interesting image return.

The dissemination activity is aimed at a broad spectrum of public and it is expected to continue even after the end of the project because the partners evaluate the objectives achieved during the project successfully.
It is important underline that dissemination activity has achieved a good response also at public level. See for instance in the magazine Horizon, 23 July 2015, Ed. BD, an interview made by Gary Finnegan.
The dissemination is generally combined with the actions of market research and marketing.
Further details are listed in deliverable D5.13:
- People interested to test the device for possible purchase
- Interested to test the device to verify if attractive to associates
- Interested to receive further information and technical details
- Other contacts in Italy and in European Countries
- Interviews at the fair VINITALY (see Annex 6 in D5.13)

The dissemination campaign was carried out successfully. The results of this campaign have been useful for planning the use and dissemination of knowledge in general about the problem of mycotoxins and in particular about the importance of controlling food and feed, as reported in D6.16 (PU deliverable) and in www.demotox.eu dissemination page.
Contamination by mycotoxin is a worldwide problem and it has increased of importance in the last years. It may be due to the climate variations that favours the growth of fungi, mainly belonging to Aspergillus and Fusarium species, also in areas where they were not previously widespread.
A specific diagram shows the relationships that are linked among them and that can exploit the results of diffusion.(Scheme of relationships coming from dissemination results).
Taking into account the various versions of the devices designed, carried out and tested, training activity has been performed during all the Project, practically without interruption. It was required a strong involvement of the Research Centres which were kept busy in the job for a long time and have spent more resource than provided (Task 5.3)
WP6 (1st -24th month) Management and coordination
The deliverables D6.14 D6.15 have been successfully submitted. D6.16 has been revised as concerns the plan for use and dissemination of knowledge. From the beginning of the project, the project coordinator and project partners have executed all technical tasks in order to fulfil the project requirements and specifications. All tasks have been properly synchronized and coordinated in order to go ahead with the development process of the project although delays have not been avoided: the planning for designing, assembling and testing of the devices was too optimistic and the risks, although indicated, was mitigated with some modification in the demonstration activities and in the use of the resources. Other internal delays raised up during the project have been properly managed and they did not affect the evolution and development process of other tasks and/or the overall project schedule. Several meetings, teleconference and telephone meetings have been organized and scheduled. A fluid communication process between the partners has been completely necessary in order to finalize all technical tasks and to fulfil the project goals as provided. The project coordinator have supported the rest of the SMEs in terms of financial reporting process and coordination activities and the Research Centres have supported the SMEs with training and assistance activity in order to complete the technical work. No consortium modifications have been done and no project extensions have been needed. However, the proposed commercial plan (see below) could interest amendment to the CA or specific new agreements among the partners. As it was previously described, all technical and management deliverables have been successfully submitted in some cases not in line with the official project deadline. The official progress reports (reporting period 1 and 2, and final progress report) and the additional progress reports requested by the project officer and reviewer have been submitted on time in line with the project deadlines.
The plan for use and dissemination of knowledge (PUDK) has been constructed starting of a detailed analysis of the foreground given by the Project.
Indeed, the heritage of knowledge for the Consortium can be indicated below.
➢ Mycotoxin effects on human and livestock health. Selection of the most dangerous mycotoxins for analysis.
➢ Impact and damage on quality of feed, food and beverage. Economic costs.
➢ Legislation applied for mycotoxin controls. Limit of detection and quantification (LOD and LOQ).
➢ Method of extraction of mycotoxins from feed and wine for analytical purposes.
➢ Methods used at the moment for controlling of the mycotoxin contamination of the feed, food and beverage. Their costs.
➢ Laboratory methods, systems on field
➢ Comparison of these methods and systems with the systems developed in the DEMOTOX Project
➢ Needs of producers of corn, wheat and wine
➢ Alternative applications of MyconixCHEMTM and MyconixBIOTM
➢ Chromatography. New aspects of the methodology
➢ Immunoassay techniques. New aspect of the methodology

Preliminarily, the management of knowledge and IPR described in CA are taken into account, as concerns foreground and background.
Then, the plan for exploitation of the results of the Project is subdivided in a specific plan of dissemination of knowledge and a specific commercial plan.
For dissemination purpose:
Diffusion of the information to a public audience about the risks of mycotoxin contamination. Diffusion to a public audience about new simple, low cost method of analysis of mycotoxins. Market analysis used to identify the most important sectors and potential buyers. Comparison with the market competitors in order to distinguish the offer.
Commercial plan and road map:
Agreement between the two producers of the devices for a limited production (100-200 systems) and for a price of sale. The market is subdivided in segments as suggested in WP4. In alternative, the producers agree to act independently, each one developing its own sale network. In this case, each producer shall pay to the other producer a percentage of the own net proceeds. This phase is assisted by CREASYS that will receive a share of X% on the net revenue. Royalties are provided for the Research Centres depending on their participation to the preparation of components (chemical kits or other). A base for define the commercial plan and hypothesis of agreement among partners, including revenues, is presented in Section “Core of 2nd periodic report” (Confidential).
A direct marketing is applied to define the real potentiality of market, identify the buyers, focusing on a short list of European countries with high production of wine, beer and cereals (Italy, Portugal, France, Spain and Germany, in particular, but also Great Britain, Russia and Poland).
Cost analysis and evaluation of the revenues can be start form the business case. The Table below (Table 5) gives idea of the revenues in the next years, starting from 200 pieces until 500 pieces sold each at €3000. The proceeds refer to one or both the devices, it does not matter which.
Year 2017 2018 2019 2020
Pieces sold (*) 200 300 400 500
Revenues(x1000) 600 900 1200 1500
(*) Cost for each device, including chemical kit, €3000
Launch into the market of the devices MyconixCHEMTM and MyconixBIOTM developed in the Project (on the basis of the package described and of possible optional components), within 2-4 years.
Scientific development of the systems of analysis in different sectors (quality of food, application to health, for instance). Scientific production for Congresses, Magazine, Workshops.
Additional benefits: reduction of environmental impact due to decreased use of chemical products (diluents and reagents) and hence less waste materials.
Dissemination of knowledge:
A synthesis of the plans of dissemination and communication is shown in table 6

Potential Impact:
4.1.4. The potential impact
(including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results (not exceeding 10 pages).
The development of an industrial prototype designed to be launched on the market has encountered several difficulties, due both to the components, not always homogeneous, both in the preparation of a compact structure and at the same time able to meet the characteristics specified in DoW.
There have been drawbacks in the technology transfer from the Research laboratory towards an industrial system and timescales in the project have not been completely met, even if the commitment in resources has not been spared by the partners. This led to the following consequences:
1. The dates indicated for the deliverables were not respected.
2. The roadmap has been changed. Initially it were planned two phases of demonstration with end users: a phase assisted by technicians and one unassisted, in which end users would have to carry out the demonstration with their samples or samples properly fortified, using devices without assistance. The first stage was performed with end users identified in Italy and Portugal. The second phase was not performed by the external organizations.
3. In place of the second phase with the end users external to the Consortium, tests have been carried out in the laboratories of AUTOMATION, LUMISENSE and an Italian qualified external laboratory (University of Milan). This modification has been justified as follows: “our clients do not possess the ability to verify if the equipment’s readings are correct or not. So they will only comment on the usability of the same after the optimization process that have been execute on these last months”.
The developed prototype devices have a high number of potential applications in a wide range of products, not only in the field of animal feed but also in the human food and in other sectors (teaching sector, for instance). Potential economic benefits and advantages both for the SME participants and for the end users can be exploited by the Consortium in the food safety market.
In conclusion, impact on the knowledge, experience and acquisition of new topics achieved by the participants with the DEMOTOX Project can be evaluated of great importance. Therefore, the potential impact can be indicated as follows:
Increased availability of new simple screening methods for detection of mycotoxin.
Reduction of environmental impact due to decreased use of chemical products (diluents and reagents) and hence less waste materials during analysis activity.
Impact on the quality of the commodities; capabilities of increasing the number of analysis of feed, food and beverage to do the screening of mycotoxins allows to give higher alimentary safety.
Impact on environment protection through the improvement of crops and of food and at the same time improved ability to intervene to reduce losses. Increasing of the number of analysis gives a greater knowledge of the food and feed contamination along the production chain and can contribute to decrease crop loss by reducing waste.
Impact on the competitiveness of the European SMEs which produce food and feed commodities, leading them towards a stronger presence within the European agro-food industry and connecting them with the European policy and European regulations. Consequently, an economic return may give further industrial growth for these Companies in term of their size and in term of employment levels. In the case of EWOS, a save of money for analysis of fish feed is expected by using the new devices to perform screening of the products.
Obviously, a good economic impact can be foreseen also for the producers of the new systems, AUTOMATION and LUMISENSE. Depending on the success of market, increase of turnover and of employment for the device producers is expected, within 4-5 years after the end of the project. In the case of CREASYS, a better impact of image and possible increase of clients can be achieved thanks to new sectors and new Companies contacted during market research and interviews made in occasion of events.
Increased possibility of exploitation of techniques for the benefit of the wine and feed producers.
Impact on the technological competitiveness of AUTOMATION and LUMISENSE. The uptake of the new technology can be influenced by different factors; in particular by its simplicity of use, low cost, portability, large spectrum of application (a number of mycotoxins more than one), capability of being used in different sectors of feed and food commodities, rules and directives strictly applied.
Potential impact on enhancing of profitability and growth performance of SMEs by combining and transferring new and existing knowledge into innovative, disruptive and competitive solutions by seizing European and global business opportunities.
Facilitation of entry of improved detection techniques in the agro-industrial sector and the market.
Social impact on human health and food safety. Screening for mycotoxin in feed and foods is a very important practice to ensure quality and safety and to eliminate or control the risk of consuming products contaminated with mycotoxins, particularly products which enter in Europe coming from other Countries.
Impact on the consumers through awareness of mycotoxins contamination in food and feed and knowledge of their toxicity on the human health.
Contribution to the sustainability of health of animal and people, through application of a higher capability of control (social impact).
Market research and direct contacts with the producers may increase likelihood of market uptake and distribution of the resulting innovations, tackling the specific challenges in a sustainable way.
Large diffusion of mycotoxins (as in summer 2012, 2013 and 2014), requires fast detection systems (short time of analysis), reliable and sensitive, low cost, user – friendly (suitable software) and possibility of use in the field (portability). At present, almost all these characteristics are satisfied by the devices designed and developed in DEMOTOX Project.
Leveraging of private investment in instrument validation, notably leverage of private co-investor and/or follow-up investments it is expected.

The various forms of described impact can be achieved through adequate dissemination activities. It is important to underline that the dissemination activities are not only focused on the interests of the end-users, but they are dedicated to all stakeholders to establish a basis for the diffusion of innovation. As already reported above, contacts have been taken with several stakeholders with the objective of disseminating potential information. A list of contacts is shown in the table below. Associations and companies involved in the demonstration tests have been selected among four targeted sectors:
- feed for fish and for animal,
- cereals,
- wine
- beer

Type of contact and country(Table 7): Association of Producers (Italy), Consortium of farmers (Italy), Wine Producers (Italy and Portugal), Producers of cereals and feed (Italy), University Research Lab (Italy), Research Institute (France), Feed producers (Norway), Bayerische Brauerbund e.V. (Germany), Agricultural Industries Confederation (UK), Brewers European Association (Belgium), Brasseurs de France (France).
The main scope is to identify the potential market, but also contribute to make some amendments/revisions/change in the current legislative framework, that might be needed to accredit the technologies.
It is reiterated here that the actions of demonstration and dissemination are interconnected between them: the objective is not only commercial (to acquire clients), but also of divulgation of new methods more practical and more simple to be used. Of course, after validation carried out in the laboratory, it will be necessary to give a stamp of certification from an International qualified Centre, as suggested also during some interviews.
During the dissemination campaign several topics have been used to give force and importance to the innovation. Among these, ease of use, reduced time of extraction, low use of chemical compounds were topics well appreciated by the contacted producers.
As regards the public dissemination activity, both devices have been presented and announced in many occasions and events, as reported in the “Dissemination” page of the website. Last public workshop, held in Rome on 20 March (see Annex 2), has been devoted to inform Italian stakeholders about the innovation, methods of analysis and about funding to the SMEs from the European Commission.
Material for the media has been properly prepared. In addition, a Press Office has been organized (see the organization chart in deliverable D5.12 and D5.13). This Office will have an important role during the commercial activity in the case of constitution of a spin off or in the case of other industrial solutions decided by the partners of the Consortium.
Table 8 is the updated Table of D5.12 shown in subsection “Public events”. In this table it is reported a list of dissemination activities which have been performed during the Project and which will be performed also after the end of the Project (see Section A).
Through the Press Office press communications have been activated. Press release are reported in D5.13 and in the public page of dissemination of the web site
Local associations of producers act as demonstrators as well as actors who spread the information: potential users and stakeholders are duly involved (Reviewer’s recommendation, Assessment Report, Ref. Ares(2014)2222178 - 04/07/2014).
Information has been disseminated to Policy makers, Scientific Community, the general public, specific group of end users as previously described.

The dissemination activity is aimed at a broad spectrum of public and it is expected to continue even after the end of the project because the partners evaluate the objectives achieved during the project successfully. The dissemination will be combined with the actions of market research and marketing.
The results of the dissemination activity, the market analysis and feedback obtained by the contacts with stakeholders encourage the SME partners, but in particular the device producers, AUTOMATION and LUMISENSE, to act in the next years for manufacturing of the MyconixCHEM and MyconixBIO devices, properly improved for launch them on the market. The results of the Project will be so exploited in the best manner.

List of Websites:
4.1.5. The address of the project public website
The address of the project public website is www.demotox.eu (www.demotox.it)
Project logo is shown in page heading
www.demotox.eu
www.demotox.it