III. Performance review report
1. Contribution of the result to the promotion policy objectives. Allergens that are not declared on food packaging pose a major risk to allergy sufferers. Some of these allergens are found in aquatic organisms such as fish, crustaceans, mollusks, and crustacean-related insects used as novel foods. Consumption can cause life-threatening reactions in affected allergy sufferers, so control laboratories and food manufacturers depend on reliable and rapid methods for detecting these species. The introduction of allergenic species can be intentional or unintentional due to carryover during transport, storage or processing along the entire value chain. Analytical traceability is of particular concern for highly processed foods, where visual inspection does not reveal the processed species. Current methods only cover single species of aquatic organisms, so a large number of methods would have to be applied to achieve high coverage. Against this background, new methods are urgently needed that allow rapid and complete detection of all commercially available species.
The results of the project fulfill several objectives of the program for innovation funding and the funding announcement "Food Production" of the German Federal Ministry of Food and Agriculture. The newly developed PCR systems for the detection of aquatic organisms and insects are versatile and increase product safety via the detection of potential allergenic species. Through close networking with the food industry (FRoSTA AG) as a collaborative partner, it was possible to demonstrate practical and product-related testing of the PCR systems. If proven in long-term testing, the "ready-to-use" plates may be of interest to biotechnology companies and thus achieve economic relevance. If the PCR systems developed are successfully transferred to standard methods, harmonization in product control will be achieved and consumer protection strengthened.
2. The scientific-technical result of the project, the achieved secondary results and the collected essential experiences. The developed real-time PCR methods close large gaps in the detection of allergenic, aquatic organisms as well as insects.
The following partial results were obtained:
A large collection of the most common commercial aquatic species and insects were authenticated as samples (reference material) and included in the sample database of the National Reference Laboratory for Animal Proteins.
New real-time PCR systems for crustaceans, mollusks, and insects were developed
in silico, and of these, twelve PCR systems were tested and validated
in vitro. Seven systems met all criteria and were also able to detect processed target species in food matrices up to 1 ppm.
The real-time PCR systems for the detection of fish and crustaceans were successfully tested in a sampling of a food production line of FRoSTA. The production line was changed from a fish/shrimp paella production to a spring vegetable ready meal production. Faint traces of crustaceans were detected even in the cleaning water between the productions.
"Ready-to-use" plates of all PCR systems for aquatic organism detection and one plate for insect detection were produced and successfully tested. The plates are available for rapid use in food control.
3. Update of the exploitation plan 3.1 Inventions/property right applications sought and property rights already granted Seq. No. 1.1 Specific exploitation: No intellectual property rights are required for the PCR arrays developed. The methods will be made freely available to potential users.
Time horizon: 2022
3.2 Economic prospects of success after the end of the project Seq. No. 2.1
Specific exploitation: As a governmental, non-profit organization,
BfRshort forGerman Federal Institute for Risk Assessment primarily pursues the following goals. To push the provision of standard methods for food control.
Time horizon: 2019-2022
Seq. No. 2.2
Specific exploitation: savings of economic resources in official control through efficient screening for unlabeled allergenic species.
Time horizon: 2022
3.3 Scientific and/or technical prospects of success after end of project Seq. No. 3.1
Specific exploitation: It is planned to summarize the results at the end of the project in the form of comprehensible consumer information. This can be realized in the form of a brochure and/or internet information on the
BfRshort forGerman Federal Institute for Risk Assessment website.
Time horizon: 2019-2023
Seq. No. 3.2
Specific exploitation: At the end of the project period, an informative workshop in presence form is planned, to which all interested parties (not only scientific experts) are to be invited.
Time horizon: 2023
Seq. No. 3.3
Specific exploitation: The collection of authentic DNA samples (dried permanent preparations) will be randomly tested annually for at least 3 years after the end of the project to maintain integrity and amplification capability. This will provide validation of the quality of the permanent preparation beyond the end of the project. An analogous durability test will also be performed on selected preloaded screening plates to document durability (application improvement).
Time horizon: 2021-2026
3.4 Scientific and economic feasibility Seq. No. 4.1
Next Phase / Next Steps: If the results of long-term monitoring are positive for the DNA collection, the principle may be useful for operators of similar collections to bypass frozen storage, which is prone to hazards and energy-intensive (improved logistics and economics).
Time horizon: 2026
Seq. No. 4.2
Next phase / next steps: Suitable real-time PCR methods will be made available to national (§-64 LFGB "Food Allergens") and international (CEN TC 275 WG 12 "Allergens") standardization bodies for conversion to standards. This will fill gaps in the official method collections.
Time horizon: 2022
Seq. No. 4.3
Next phase / next steps: The extent to which broader availability of "ready-to-use" PCR microplates can be achieved and whether transfer to other areas (authenticity) makes sense should be examined. Depending on resources, this could be done in-house or subcontracted to third parties.
Time horizon: 2022
4. Work that did not lead to a solution The amplification temperatures of the real-time PCR systems developed ranged from 55 to 60°C. Therefore, a 384-well PCR microtiter plate was not implemented for the "ready-to-use" plates as originally planned, since this would require the systems to all be used at the same temperature. The systems were split between two 96-well PCR microtiter plates, resulting in one plate with purely aquatic organisms and one insect plate for "novel foods". The advantage of 96-well PCR microplates is that they are the most common model in control analysis, can be easily replicated by third-party vendors, and still have enough space for 6 samples. We tested a 384-well PCR plate so that the number of samples could be quadrupled.
5. Presentation possibilities for potential users The
BfRshort forGerman Federal Institute for Risk Assessment is a guest member of the §-64 LFGB working group "Food Allergens", where new methods are presented and included in the "Official Collection of Test Methods". The already published real-time PCR method for the detection of crustaceans has been proposed for a pre-ring trial, which is planned for 2023.
6. Adherence to cost and time schedule The project could be realized within the approved budget, there were only some delays due to the Corona crisis, therefore a cost-neutral extension of half a year was requested and approved.