Fermentative Gewinnung von Stoffe mit Hilfe von gentechnisch veränderten Mikroorganismen (GMMO)
rDNA in Fermentationsprodukten - Publikationen, Stellungnahmen, Regulation zu rDNA
Entspricht das Vorkommen von rDNA in Fermentationsprodukten der Präsenz von lebenden GVO?
EU-Kommission, EU-Parlament und Umfeld
Tom Vandenkendelaere (PPE): Exclude microbial fermentation products containing GMO rDNA residues from GMO
legislation (9.4.2022)
Question for written answer E-001419/2022 to the Commission Rule 138
https://www.europarl.europa.eu/doceo/document/E-9-2022-001419_EN.html
Answer given by Ms Kyriakides on behalf of the European Commission (23.5.2022)
https://www.europarl.europa.eu/doceo/document/E-9-2022-001419-ASW_EN.htm
SCoPAFF: Standing Committee on Plants, Animals, Food and Feed Section Genetically Modified Food and Feed - 20 September 2021
Diskussionspunkt A.07
EURL: European Union Reference Laboratory for Genetically Modified Food and Feed (EURL GMFF)
https://gmo-crl.jrc.ec.europa.eu/
ENGL: The European Network of GMO Laboratories (ENGL) is a consortium of official enforcement laboratories designated by the
EU Member States plus Norway, Switzerland and Turkey.
https://gmo-crl.jrc.ec.europa.eu/ENGLabs
Meeting Reports
https://gmo-crl.jrc.ec.europa.eu/ENGLabs#inline-nav-engl-work
42nd ENGL Steering Committee Meeting - Meeting report, 24 February 2022
https://gmo-crl.jrc.ec.europa.eu/ENGL/docs/42nd%20ENGLSC_Report.pdf
Our publications
https://gmo-crl.jrc.ec.europa.eu/publications
Sciensano ist am 01.08.2024 zum Referenzlaborator der Europäischen Union für Lebensmittelverbesserungsmittel ernannt worden.
►Durchführungsbeschluss (EU) 2024/1941 der Kommission vom 22. Juli 2024zur Benennung eines Referenzlaboratoriums der
Europäischen Union für Stoffe zur Verbesserung von Lebensmitteln im Einklang mit der Verordnung (EU) 2017/625 des Europäischen Parlaments und des Rates. ABl L 24.07.2024
(siehe hierzu auch die Publikationen von Faiture et al.)
Relevante gesetzliche Regelungen - Richtlinien und Verordnungen
► RL 2001/18/EG : Richtlinie 2001/18/EG des Europäischen Parlaments und des Rates vom 12. März 2001 über die absichtliche
Freisetzung genetisch veränderter Organismen in die Umwelt und zur Aufhebung der Richtlinie 90/220/EWG des Rates. ABl. L 106, 1-37
► Richtlinie 2009/41/EG des Europäischen Parlamentes und des Rates vom 6. Mai 2009 über die Anwendung genetisch veränderter
Mikroorganismen in geschlossenen Systemen. Abl. L 25, 75-97 vom 21.05.2009
► Verordnung (EG) Nr. 1829/2003 des Europäischen Parlaments und des Rates vom 22. September 2003 über genetisch veränderte
Lebensmittel und Futtermittel. ABl L 268, 1-23
Siehe für: Lebensmittel Artikel 3 und teilweise Artikel 4; Futtermittel Artikel 15
Es wird nur Bezug auf GVO genommen, nicht auf rDNA. GVO bezieht sich entsprechend der Freisetzungsrichtline auf den lebenden, vermehrungsfähigen Organismus. Aber die Verordnung hat Fragen aufgeworfen, u.a. ob Fermentationsprodukte die den GMMO nicht enthalten unter die Verordnung fallen. Unterscheidung zwischen „hergestellt aus und hergestellt mit….“ (siehe unten)
► Verordnung (EG) Nr. 1830/2003 des Europäischen Parlaments und des Rates vom 22. September 2003 über die Rückverfolgbarkeit
und Kennzeichnung von genetisch veränderten Organismen und über die Rückverfolgbarkeit von aus genetisch veränderten Organismen hergestellten Lebensmitteln und Futtermitteln sowie zur Änderung der Richtlinie 2001/18/EG. ABl. L. 268, 24-28
► SCoPAFF: Standing Committee on the food chain and animal health – Summary record of the 3rd meeting, 24 September 2004
1. Implementation of Regulation (EC) N° 1829/2003
“Food and feed (including food and feed ingredients such as additives, flavourings and vitamins) produced by fermentation using a genetically modified micro-organism (GMM) which is kept under contained conditions and is not present in the final product are not included in the scope of Regulation (EC) No 1829/2003. These food and feed have to be considered as having been produced with the GMM, rather than from the GMM. Food and feed (including food and feed ingredients such as additives, flavourings and
vitamins) produced by fermentation using a genetically modified micro-organism (GMM) which is present in the final product, totally or partially, whether alive or not, are included in the scope of Regulation (EC) No 1829/2003, in regard of both authorisation and labelling.”
► Report from the Commission to the Council and the EU-Parliament on the implementation of Regulation (EC) No 1829/2003 of the European Parliament and of the Council on genetically modified food and feed
10. Status of food or feed produced by fermentation using genetically modified micro-organisms
not present in the final product .............23 10.1. Background ............. 23
10.2. Clarification of the status of food or feed produced by fermentation using genetically modified
micro-organisms not present in the final product ............. 24
10.3. Application of the clarification to food products and processing aids... ............. 24
10.4. Application of the clarification to feed products and processing aids ............. 25
10.5. Guidelines on the Safety assessment of food or feed produced by fermentation using genetically
modified micro-organisms. ............. 26
Food Improvement Agent Package - Das Regelwerk für Aromen, Zusatzstoffe und Enzyme
► Verordnung (EG) Nr. 1331/2008 des Europäischen Parlaments und des Rates vom 16. Dezember 2008 über ein einheitliches
Zulassungsverfahren für Lebensmittelzusatzstoffe, -enzyme und –aromen. ABl. L 354, 1-5
► Verordnung (EG) Nr. 1332/2008 des Europäischen Parlaments und des Rates vom 16. Dezember 2008 über Lebensmittelenzyme
und zur Änderung der Richtlinie 83/417/EWG des Rates, der Verordnung (EG) Nr. 1493/1999 des Rates. ABl. L 354, S. 7-15
► Verordnung (EG) Nr. 1333/2008 des Europäischen Parlaments und des Rates vom 16. Dezember 2008 über Lebensmittel-
zusatzstoffe. ABl. L 354, 16-33
► Verordnung (EG) Nr. 1334/2008 des Europäischen Parlaments und des Rates vom 16. Dezember 2008 über Aromen und
Lebensmittelzutaten mit aromatisierten Eigenschaften. ABl. L 354, 34-50 Neuer Text
EFSA:
CEP Panel (2021): Scientific Guidance for the submission of dossiers on Food Enzymes. EFSA Journal 19 (10):6851, 37 pp.
https://doi.org/10.2903/j.efsa.2021.6851
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2021.6851
Explanatory Note for the Guidance of the Scientific Panel of Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) on the Submission of a Dossier on Food Enzymes. EFSA Supporting Publication 2014; 11(11): EN-689. 22 pp. doi:10.2903/sp.efsa.2014.EN-689
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/sp.efsa.2014.EN-689
GMO-Panel (2011): SCIENTIFIC OPINION- Guidance on the risk assessment of genetically modified microorganisms and
their products intended for food and feed use
https://efsa.onlinelibrary.wiley.com/doi/pdf/10.2903/j.efsa.2011.2193
CEF Panel (2009) Guidance of EFSA prepared by the Scientific Panel of Food Contact Material, Enzymes, Flavourings and Processing
Aids on the Submission of a Dossier on Food Enzymes. EFSA Journal7 (5):1305, 26 pp.
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2009.1305
Die Leitlinie von 2021 ist gültig und anzuwenden; die beiden anderen sollen nur die Weiterentwicklung aufzeigen.
In Leitlinie von 2021 ist 1.3.4.1. Viable cells of the production strain und 1.3.4.2. DNA from the production strain zu beachten. "For the purpose of this assessment, the applicant should investigate whether the target DNA is detected in analyses having a LoD of 10 ng of DNA per g or mL of product or lower."
EFSA meeting, 20 June 2023: GUIDANCE ON THE CHARACTERISATION OF MICROORGANISMS USED AS FEED ADDITIVES OR
AS PRODUCTION ORGANISMS - Eine Präsentation
https://www.efsa.europa.eu/sites/default/files/2023-06/20230620-industry-presentation.pdf
Inter-association meeting DNA and fermentation products (Präsentation)
https://fefac.eu/wp-content/uploads/2021/07/21_DOC_74.pdf
Publikationen, Statements zu rDNA in Fermentationsprodukten
Pelzer, S., Lensch, A. (2024): Grenzwert für DNA in Fermentationsprodukten schadet Industrie und Akademie.
Biospektrum 30, 615 | https://doi.org/10.1007/s12268-024-2318-3
https://link.springer.com/article/10.1007/s12268-024-2318-3
DIB (2024): Bioökonomie: Praxisnahe Gesetzgebung beibehalten
https://www.vci.de/services/publikationen/biotech-brief/biooekonomie.jsp
Dederer H.-G. (2021): rDNA Traces in Fermentation Products Using Genetically Modified Microorganisms (GMMs),
Zeitschrift für Stoffrecht 18 (3), 135 - 147 | https://doi.org/10.21552/stoffr/2021/3/6
Certain products such as amino acids, flavourings, oligosaccharides, organic acids, or vitamins are obtained by fermentation using genetically modified microorganisms (GMMs). Such fermentation products may be used as or in food and feed. Although the GMMs are separated from the fermentation products during downstream processing these products may, nevertheless, contain traces of rDNA originating from the GMMs. The European Commission holds the view that fermentation products obtained by using GMMs are subject to Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed to the extent that rDNA is still present in the fermentation product irrespective of the amount of rDNA sequences. However, it follows from the travaux préparatoires as well as early discussions on the scope of Regulation (EC) No 1829/2003 starting immediately after its entry into force that it was neither designed nor framed to be applicable to fermentation products obtained by the use of GMMs. Accordingly, Regulation (EC) No 1829/2003 cannot be considered to be fit for purpose as regards regulation of such products. In particular, it is clear from the regulation’s wording and context that it does not apply to food or feed products obtained by fermentation using GMMs if the GMMs have been removed during downstream processing. In this case, the GMMs are mere ‘processing aids’ within the meaning of Recital 16, sentences 3 and 4, of Regulation (EC) No 1829/2003. Therefore, such food or feed products obtained by fermentation using GMMs are excluded from the scope of the regulation since, first, these products are not produced ‘from’ but produced ‘with’ GMMs (cf. Artt. 2(6), (7) and (10), 3(1)(c), 15(1)(c) of Regulation (EC) No 1829/2003 as construed in light of Recital 16, sentences 1, 3 and 4) and, second, the GMMs are not ‘source material’ of the fermentation products (cf. Art. 2(8) and (9), Art. 3(1)(a), Art. 15(1)(a) of Regulation (EC) No 1829/2003 as construed in light of Recital 16, sentences 1, 3 and 4). As GMMs are ‘processing aids’ within the meaning of sentences 3 and 4 of Recital 16 and the fermentation products are, therefore, produced ‘with’ the GMMs within the meaning of sentence 1 of Recital 16, sentence 2 of the recital has, logically, no relevance as regards the distinction between food or feed produced ‘from’ or ‘with’ GMMs. rDNA traces in fermentation products obtained by the use of GMMs are not ‘ingredients’ either. Rather, they constitute mere ‘residues’. Any health safety concerns related to the presence of rDNA traces are addressed by other Union legislation, e.g., on food additives, food enzymes and food flavourings or feed additives and other feed materials, respectively, or, as the case may be, on novel foods.
https://stoffr.lexxion.eu/data/article/17593/pdf/stoffr_2021_03-008.pdf
Lensch A., Duwenig E., Dederer H.-G., Kärenlampi S.O., Custers R., Borg A., Wyss M. (2022): Recombinant DNA in fermentation
products is of no regulatory relevance. Food Control | https://doi.org/10.1016/j.foodcont.2022.109170
A large variety of fermentation products are used in food and feed production, but also in other industries, and many of these products are produced with genetically modified microorganisms (GMMs). In food and feed production, prominent examples are amino acids, vitamins, food and feed enzymes, colorants, non-caloric sweeteners, human milk oligosaccharides, or vegan alternatives of dairy, egg and meat products.
From a regulatory perspective, fermentation products are typically produced under containment. This means that premises, equipment and work processes need to be designed to prevent or at least minimize release of GMMs into the environment. The fermentation products themselves should not contain any live cells of the GMM. Over the past years, there have been concerning developments, particularly in the European Union, stipulating that also absence of recombinant DNA might be interpreted as a regulatory requirement for fermentation products produced with GMMs.
In this paper, we (i) attempt to place these developments into the historical context, (ii) sketch the potential negative repercussions for the food and feed industries, (iii) elaborate on the safety of recombinant DNA, and (iv) postulate that recombinant DNA should remain an integral part of the safety assessment of fermentation products but should not be misconstrued as a criterion for regulatory classification of products of biotechnology.
https://www.sciencedirect.com/science/article/pii/S0956713522003632
Wesseler J., Kleter G., Meulenbroek M., Purnhagen K.P. (2022): EU regulation of genetically modified microorganisms in light
of new policy developments: Possible implications for EU bioeconomy investments. Appl Econ Perspect Policy. 1–21.
Many developments in the bioeconomy depend on the use of genetically modified microorganisms (GMMs). GMMs are used in bioreactors to convert biomass into food, feed, and energy products. The recent judgment by the Court of Justice of the European Union on gene editing technologies has affected the use of GMMs. A heated debate has started on whether and under what circumstances GMMs should be considered genetically modified organisms. This kind of decision is extremely relevant, as it will have a strong effect on the innovation of sustainable supply chains in the bioeconomy. The question has been raised as to whether the regulatory policies on GMMs can be justified from a sustainability perspetive and, in particular, whether they do not endanger the European Green Deal, the flagship policy strategy of the new European Commission under Ursula von der Leyen. This contribution will first pro ide an overview of GMMs and their importance for the development of the bioeconomy, followed by a theoretical framework for assessing investments in GMMs. The third part of the article includes a discussion of our scenarios for regulating GMMs in the future, derived from the EU legal environment. The potential implications of the scenarios are assessed by linking them with the benefits and costs of investments in GMMs, following a modified version of the model presented in Purnhagen and Wesseler (2019). The results show that reforms based on the current EU legal environment do not look very promising to further support the use of GMMs. This has important implications for reaching the objectives of the Green Deal, as more radical legal changes are needed for the success of the initiative.
BioSafe: The EU's stance on genetically modified microorganisms (GMMs)
https://www.biosafe.fi/insight/the-eus-stance-on-genetically-modified-microorganisms-gmms
hier auch der "Schwellenwert" von 10 ng rDNA/1g Produkt erwähnt!
BiosSafe: Mistakes to avoid when analysing recombinant DNA in food products
https://www.biosafe.fi/insight/mistakes-to-avoid-when-analysing-recombinant-dna-in-food-products
Precup G, Marini E, Zakidou P, Beneventi E, (2024): Novel foods, food enzymes, and food additives derived from food
by-products of plant or animal origin: principles and overview of the EFSA safety assessment. Front. Nutr. 11:1390734 doi: 10.3389/fnut.2024.1390734
The European Union (EU) is committed to transitioning toward a circular economy model, with food waste being one of the areas to be targeted. To close the loop of food waste generated during food processing and discarded at the retail or consumption phases, research and innovation parties proposed to valorize agro-food by-products to produce novel foods and food improvement agents (food additives, food enzymes, and food flavorings). In the EU, the authorization of such novel foods and food improvement agents is governed by different regulatory frameworks. A centralized safety assessment by the European Food Safety Authority (EFSA) is the prerequisite for their authorization through the so-called Union Lists. Up to December 2023, EFSA published 45 scientific opinions on the safety of novel foods, food enzymes, and food additives derived from by-products of plant and animal origin. The current study illustrates examples of these by-products for the production of novel foods or food improvement agents and the data requirements behind their respective safety assessments conducted by EFSA. In this review, applications on novel foods, food enzymes, and food additives received by EFSA were screened and analyzed to find the common scientific requirements and differences in terms of the safety evaluation of such products. Various by-products (i.e., corncobs, coffee husks, spent grains of barley and rice, grape pomace, pumpkin peels, bovine whey, eggshells, shrimp heads, and animal organs or tissues) were described in the applications as being processed (extraction, physical treatments, and chemical and enzymatic reactions) to obtain novel foods and food improvement agents. The heterogeneity and complexity of these products emphasize the challenge of their safety assessment, depending on the characteristics of each product. However, as this study shows, the scientific requirements underpinning their safety do not differ substantially in the different regulated product areas considered, with similar information needed to assess their safety in terms of identity, production process, compositional characterization, proposed/intended uses and exposure assessment, toxicological information, and allergenicity data. Additional nutritional information and data on the history of use are required in the case of novel foods.
https://www.frontiersin.org/journals/nutrition/articles/10.3389/fnut.2024.1390734/full
Nachweis von rDNA in Fermentationsprodukten
EMA and EFSA: Expectations on Analytical GMO Detection Limits in Biotechnology Products (2023)
De Keersmaecker, S.C., Kok, E., Roosens, N., Zaoui, X. et al. (2024): Sequencing strategies for the traceability of GMOs -
methods and related quality aspects. Publications Office of the European Union, Luxembourg, | https://data.europa.eu/doi/10.2760/890142, JRC137607
Deckers M.(2022): Strategies to control the presence of microbial impurities, including genetically modified
microorganisms, in food enzymes. Ghent University. Faculty of Pharmaceutical Sciences, Ghent, Belgium.
Enzymes have been used for decades in various food processes. They are increasingly being produced by fermentation using micro-organisms, including genetically modified micro-organisms (GMM). The (EC) No 1332/2008 regulation requires that food enzymes (FE) sold on the EU market follow a safety evaluation in order to be approved for sale and one of the investigated criteria during the evaluation is the absence of the producer organism, both viable cells and associated DNA. However, no detection methods were available to investigate FE preparations regarding the presence of microbial impurities, including FE producing micro-organisms. Additionally, most of the information needed for the development of appropriate detection methods is submitted confidentially. Lastly, since no investigations had been performed on EU commercialized FE, the competent authorities were not able to determine whether or not such microbial impurities could occur. This PhD thesis aimed to contribute to the development of a control strategy targeting microbial impurities related to producing organisms in FE preparations and to obtain an overview of these potential contaminations. First, public information, dispersed in many documents, was collected, structured and analyzed. The collected data was structured and gathered into an open-access web application. Second, generic methods were developed for the detection of FE producing organisms, including bacteria and fungi. Combined to a recently available GMM detection strategy, these generic methods were applied on a representative collection of FE sold on the EU market. This pilot monitoring allowed to verify the applicability of the detection strategies on FE and to provide a first picture of the current situation related to the contamination of FE with their producing organism. Furthermore, the generated results allowed to identify potential needs to guarantee the safety and traceability of the food chain.
Fraiture M.-A., Gobbo A., Guillitte, C. Marchesi E., Verginelli D. et al. (2024): Pilot market surveillance of GMM contaminations in
alpha-amylase food enzyme products: A detection strategy strengthened by a newly developed qPCR method targeting a GM Bacillus licheniformis producing alpha-amylase. Food Chemistry: Molecular Sciences 8 , 100186 | https://doi.org/10.1016/j.fochms.2023.100186
Using high-throughput metagenomics on commercial microbial fermentation products, DNA from a new unauthorized genetically modified microorganism (GMM), namely the GM B. licheniformis strain producing alpha-amylase (GMM alpha-amylase2), was recently discovered and characterized. On this basis, a new qPCR method targeting an unnatural association of sequences specific to the GMM alpha-amylase2 strain was designed and developed in this study, allowing to strengthen the current GMM detection strategy. The performance of the newly developed qPCR method was assessed for its specificity and sensitivity to comply with the minimum performance requirements established by the European Network of GMO Laboratories for GMO analysis. Moreover, the transferability of the in house validated qPCR method was demonstrated. Finally, its applicability was confirmed by a pilot market surveillance of GMM contaminations conducted for the first time on 40 alpha-amylase food enzyme products labelled as containing alpha-amylase. This pilot market surveillance allowed also to highlight numerous contaminations with GMM alpha-amylase2, including frequent cross-contaminations with other GMM strains previously characterized. In addition, the presence of full-length AMR genes, raising health concerns, was also reported.
https://www.sciencedirect.com/science/article/pii/S2666566223000266
Fraiture M.-A., Gobbo A., Papazova N., Roosens N.H.C. (2022): Development of a Taxon-Specific Real-Time PCR Method
Targeting the Bacillus subtilis Group to Strengthen the Control of Genetically Modified Bacteria in Fermentation Products. Fermentation 8 (2), 78 | https://doi.org/10.3390/fermentation8020078
Most of the bacteria that are used to produce fermentation products, such as enzymes, additives and flavorings, belong to the Bacillus subtilis group. Recently, unexpected contaminations with unauthorized genetically modified (GM) bacteria (viable cells and associated DNA) that were carrying antimicrobial resistance (AMR) genes was noticed in several microbial fermentation products that have been commercialized on the food and feed market. These contaminations consisted of GM Bacillus species belonging to the B. subtilis group. In order to screen for the potential presence of such contaminations, in this study we have developed a new real-time PCR method targeting the B. subtilis group, including B. subtilis, B. licheniformis, B. amyloliquefaciens and B. velezensis. The method’s performance was successfully assessed as specific and sensitive, complying with the Minimum Performance Requirements for Analytical Methods of GMO Testing that is used as a standard by the GMO enforcement laboratories. The method’s applicability was also tested on 25 commercial microbial fermentation products. In addition, this method was developed to be compatible with the PCR-based strategy that was recently developed for the detection of unauthorized GM bacteria. This taxon-specific method allows the strengthening of the set of screening markers that are targeting key sequences that are frequently found in GM bacteria (AMR genes and shuttle vector), reinforcing control over the food and feed chain in order to guarantee its safety and traceability.
https://www.mdpi.com/2311-5637/8/2/78
Fraiture, MA., Deckers, M., Papazova, N. et al. (2020): Strategy to Detect Genetically Modified Bacteria Carrying Tetracycline
Resistance Gene in Fermentation Products. Food Anal. Methods 13, 1929–1937 (2020). https://doi.org/10.1007/s12161-020-01803-6
Unexpected contaminations of unauthorized genetically modified microorganisms (GMM) harbouring antimicrobial resistance (AMR) genes in food and feed enzymes, additives and flavourings commercialized on the European market have recently alerted the competent authorities regarding the food and feed safety. At the control level, we have therefore proposed a PCR-based strategy as first line screening targeting GMM carrying AMR genes in order to help enforcement laboratories. The potential presence of frequently used AMR genes is first investigated, using real-time PCR. In case of a suspicious matrix, the full-length of the detected AMR genes is then determined, using conventional PCR followed by Sanger sequencing, allowing to support the competent authorities in their evaluation related to potential health risks. In this study, PCR methods targeting an additional key AMR gene, being the tet-L gene (GenBank: D00946.1) conferring a resistance to tetracycline, were developed and successfully assessed in terms of specificity, sensitivity and applicability. In integrating these PCR methods, the proposed PCR-based strategy, initially targeting two key AMR genes conferring a resistance to chloramphenicol (GenBank: NC_002013.1) and kanamycin (GenBank: M19465.1), is consequently strengthened, allowing the coverage of a larger spectrum of potential GMM contaminations in microbial fermentation products.
https://link.springer.com/article/10.1007/s12161-020-01803-6
Fraiture, M.A., Bogaerts, B., Winand, R. et al. (2020): Identification of an unauthorized genetically modified bacteria in food
enzyme through whole-genome sequencing. Sci Rep 10, 7094 | https://doi.org/10.1038/s41598-020-63987-5
Recently, the unexpected presence of a viable unauthorized genetically modified bacterium in a commercialized food enzyme (protease) product originating from a microbial fermentation process has been notified at the European level (RASFF 2019.3332). This finding was made possible thanks to the use of the next-generation sequencing technology, as reported in this study. Whole-genome sequencing was used to characterize the genetic modification comprising a sequence from the pUB110 shuttle vector (GenBank: M19465.1), harbouring antimicrobial resistance genes conferring a resistance to kanamycine, neomycin and bleomycin, flanked on each side by a sequence coding for a protease (GenBank: WP_032874795.1). In addition, based on these data, two real-time PCR methods, that can be used by enforcement laboratories, specific to this unauthorized genetically modified bacterium were developed and validated. The present study emphasizes the key role that whole-genome sequencing can take for detection of unknown and unauthorized genetically modified microorganisms in commercialized microbial fermentation products intended for the food and feed chain. Moreover, current issues encountered by the Competent Authorities and enforcement laboratories with such unexpected contaminations and the importance of performing official controls were highlighted.
https://www.nature.com/articles/s41598-020-63987-5
D’aes, J.; Fraiture, M.-A.; Bogaerts, B.; De Keersmaecker, S.C.J.; Roosens, N.H.C.; Vanneste, K. (2021): Characterization of
Genetically Modified Microorganisms Using Short- and Long-Read Whole-Genome Sequencing Reveals Contaminations of Related Origin in Multiple Commercial Food Enzyme Products. Foods, 10, 2637 | https://doi.org/10.3390/foods10112637
Despite their presence being unauthorized on the European market, contaminations with genetically modified (GM) microorganisms have repeatedly been reported in diverse commercial microbial fermentation produce types. Several of these contaminations are related to a GM Bacillus velezensis used to synthesize a food enzyme protease, for which genomic characterization remains currently incomplete, and it is unknown whether these contaminations have a common origin. In this study, GM B. velezensis isolates from multiple food enzyme products were characterized by short- and long-read whole-genome sequencing (WGS), demonstrating that they harbor a free recombinant pUB110-derived plasmid carrying antimicrobial resistance genes. Additionally, single-nucleotide polymorphism (SNP) and whole-genome based comparative analyses showed that the isolates likely originate from the same parental GM strain. This study highlights the added value of a hybrid WGS approach for accurate genomic characterization of GMM (e.g., genomic location of the transgenic construct), and of SNP-based phylogenomic analysis for source-tracking of GMM.
https://www.mdpi.com/2304-8158/10/11/2637
Benini C., Borg A., Downes C., Seifert J. (2021): RE: Letter to the Editor – Analytical strategy for EU control laboratories to
detect the presence of rDNA. Food Chemistry 350, 128703 | https://doi.org/10.1016/j.foodchem.2020.128703
https://www.sciencedirect.com/science/article/abs/pii/S0308814620325656?via%3Dihub
Beispiel: rDNA und GMMO im Futtermittelzusatzstoff Vitamin B2
Fraiture M.-A., Joly L., Vandermassen E., Delvoye M.: et al. (2021): Retrospective survey of unauthorized genetically modified
bacteria harbouring antimicrobial resistance genes in feed additive vitamin B2 commercialized in Belgium: Food Control 119, 107476 | https://doi.org/10.1016/j.foodcont.2020.107476
In Belgium, an official control plan was established in 2016 to detect the potential presence of an unauthorized genetically modified (GM) Bacillus subtilis RASFF2014.1249 strain in commercialized feed additive vitamin B2 products. To this end, two real-time PCR markers specific to this unauthorized genetically modified microorganism (GMM), named UGMVit-B2 and 558, were used. In the present study, the first four-year results from 67 feed additive vitamin B2 samples from the official control are presented. It includes 5 samples positive for real-time PCR methods specific to the unauthorized GM B. subtilis RASFF2014.1249 strain and has led to the RASFF2018.2755 and RASFF2019.3216 notifications. Moreover, a retrospective study using the same feed additive vitamin B2 samples was performed, allowing to provide a first picture of GM bacterial contaminations. It consisted in a first-line screening strategy gathering available PCR-based methods targeting both the B. subtilis species, frequently used to produce vitamin B2, and a set of antimicrobial resistance (AMR) genes commonly harboured as selection marker by GM bacteria used to produce microbial fermentation products. On this basis, suspicious samples contaminated with additional unknown GM bacterial strains as well as potential health and environmental risks related to the unexpected presence of full-length AMR genes could be highlighted. In addition, the possible complementary use of additional data, like chloramphenicol presence and DNA concentration, as indicators for GMM contaminations was assessed. Based on results generated in the present study, the relevance to use the proposed first-line screening strategy supplemented by indicators in order to strengthen the current control strategy was emphasized.
https://www.sciencedirect.com/science/article/pii/S0956713520303923
EFSA
FEEDAP Panel (2014): Scientific Opinion on the safety and efficacy of vitamin B2 (80 %) as riboflavin produced by
Bacillus subtilis for all animal species, based on a dossier submitted by VITAC EEIG. EFSA Journal 2014; 12(1):3531, 21 pp. doi:10.2903/j.efsa.2014.3531
„The use of the additive in animal nutrition does not pose a risk to the environment. The additive is regarded as an effective source of riboflavin in covering the animal’s requirement when administered orally.”
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2014.3531
FEEDAP Panel (2018): Scientific Opinion on the safety of vitamin B2 (80%) as riboflavin produced by Bacillus subtilis
KCCM-10445 for all animal species. EFSA Journal 2018;16(3): 5223, 8 pp. https://doi.org/10.2903/j.efsa.2018.5223
“The analysed samples contained DNA belonging to the production strain, including the genetic modification. Moreover, one of the samples contained viable cells from the production strain. Because the production strain carries antimicrobial resistance genes introduced by the genetic modification, the FEEDAP Panel considers that Riboflavin (80%) poses a risk for the spread of viable cells and DNA of a genetically modified strain-harbouring genes coding for resistance to antimicrobials.”
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2018.5223
Durchführungsverordnung (EU) 2018/1254 der Kommission vom 19. September 2018 zur Verweigerung der Zulassung von
Riboflavin (80 %), hergestellt aus Bacillus subtilis KCCM-10445, als Futtermittelzusatzstoff in der Funktionsgruppe Vitamine, Provitamine und chemisch definierte Stoffe mit ähnlicher Wirkung. ABl L 237, 5-7 vom 20.09.2018
Artikel 1 Verweigerung der Zulassung
Die Zulassung von Riboflavin (80 %), hergestellt aus Bacillus subtilis KCCM-10445, als Futtermittelzusatzstoff in der Funktionsgruppe Vitamine, Provitamine und chemisch definierte Stoffe mit ähnlicher Wirkung (im Folgenden der „Zusatzstoff“) wird verweigert.
https://eur-lex.europa.eu/legal-content/DE/TXT/PDF/?uri=CELEX:32018R1254
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Barbau-Piednoir, E., De Keersmaecker, S.C.J., Delvoye, M. et al. (2015): Use of next generation sequencing data to develop a
qPCR method for specific detection of EU-unauthorized genetically modified Bacillus subtilis overproducing riboflavin. BMC Biotechnol 15, 103 | https://doi.org/10.1186/s12896-015-0216-y
Background: Recently, the presence of an unauthorized genetically modified (GM) Bacillus subtilis bacterium overproducing vitamin B2 in a feed additive was notified by the Rapid Alert System for Food and Feed (RASFF). This has demonstrated that a contamination by a GM micro-organism (GMM) may occur in feed additives and has confronted for the first time,the enforcement laboratories with this type of RASFF. As no sequence information of this GMM nor any specific detection or identification method was available, Next GenerationSequencing (NGS) was used to generate sequence information. However, NGS data analysis often requires appropriate tools, involving bioinformatics expertise which is not alwayspresent in the average enforcement laboratory. This hampers the use of this technology to rapidly obtain critical sequence information in order to be able to develop a specific qPCRdetection method.
Methods: Data generated by NGS were exploited using a simple BLAST approach. A TaqMan® qPCR method was developed and tested on isolated bacterial strains and on the feed additive directly.
Results: In this study, a very simple strategy based on the common BLAST tools that can be used by any enforcement lab without profound bioinformatics expertise, was successfully used toanalyse the B. subtilis data generated by NGS. The results were used to design and assess a new TaqMan® qPCR method, specifically detecting this GM vitamin B2 overproducing bacterium. The method complies with EU critical performance parameters for specificity, sensitivity, PCR efficiency and repeatability. The VitB2-UGM method also could detect the B. subtilis strain in genomic DNA extracted from the feed additive, without prior culturing step.
Conclusions: The proposed method, provides a crucial tool for specifically and rapidly identifying this unauthorized GM bacterium in food and feed additives by enforcement laboratories. Moreover, this work can be seen as a case study to substantiate how the use of NGS data can offer an added value to easily gain access to sequence information needed to develop qPCR methods to detect unknown andunauthorized GMO in food and feed.
https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-015-0216-y
Paracchini V., Petrillo M., Reiting R., Angers-Loustau A. et al. (2017): Molecular characterization of an unauthorized genetically
modified Bacillus subtilis production strain identified in a vitamin B2 feed additive. Food Chemistry 230, 681-689 | https://doi.org/10.1016/j.foodchem.2017.03.042
Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive. A vitamin B2 product (80% feed grade) imported from China was analysed. Viable B. subtilis cells were identified and DNAs of two bacterial isolates (LHL and LGL) were subjected to three whole genome sequencing (WGS) runs with different devices (MiSeq, 454 or HiSeq system). WGS data revealed the integration of a chloramphenicol resistance gene, the deletion of the endogenous riboflavin (rib) operon and presence of four putative plasmids harbouring rib operons. Event- and construct-specific real-time PCR methods for detection of the GM strain and its putative plasmids in food and feed products have been developed.
https://www.sciencedirect.com/science/article/pii/S0308814617304193?via%3Dihub
Park S., Kim T., Lee S., Kim P. (2024): Safety Regulations of Edible Ingredients Derived from Corynebacterium glutamicum.
Food Sci & Nutri Tech 2024, 9(1): 000328
Corynebacterium glutamicum has been researched and developed as a strain that produces various edible ingredients, starting with glutamic acid. Due to environmental pollution and food security issues, studies on using C. glutamicum as a single-cell protein (SCP) are emerging. For the past 20 years, cases valid as safe among edible ingredients derived from C. glutamicum by the European Food Safety Authority (EFSA) or U.S. Food and Drug Administration (FDA) were L-glutamic acid, L-lysine, L-arginine, L-valine, L-isoleucine, L-histidine, L-threonine, L-methionine, L-tryptophan, L-glutamine, 2′-fucosyllactose, corn syrup fermentation product, and D-psicose 3-epimerase. According to validation, it is recommended that the final product should be free of viable cells and recombinant DNA of the production strain. Although there is a possibility that viable cells may be present in the final product, it can be considered safe if the strain qualifies for Qualified Presumption of Safety (QPS) or Generally Recognized as Safe (GRAS). Even if there is a possibility that recombinant DNA may be present in the final product,it can be considered safe as long as it is not a gene of concern for antibiotic resistance, toxicity, or pathogenicity. This review provides insights for future safety validation of edible ingredients derived from C. glutamicum, including SCPs.
https://medwinpublishers.com/FSNT/safety-regulations-of-edible-ingredients-derived-from-corynebacterium-glutamicum.pdf
EFSA FEEDAP Panel (2023): Scientific opinion on the presence of DNA in the feed additive consisting of l-isoleucine
produced by Corynebacterium glutamicum KCCM 80185 for all animal species (CJ Europe GmbH). EFSA Journal 2023; 21(4):7957, 5 pp. https://doi.org/10.2903/j.efsa.2023.7957
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2023.7957
Dederer H.-G., Hamburger D. (2022): Are genome-edited micro-organisms covered by Directive 2009/41/EC?—Implications
of the CJEU’s judgment in the case C-528/16 for the contained use of genome-edited micro-organisms.
Journal of Law and the Biosciences 9 (1), lsab033, | https://doi.org/10.1093/jlb/lsab033
In its judgement of July 25, 2018, the Court of Justice of the European Union (CJEU) in the case C-528/16, Confédération paysanne and Others, held that organisms obtained by techniques of mutagenesis are ‘genetically modified organisms’ (GMOs). It follows from the Court’s reasoning that genome-edited organisms, ie organisms resulting from techniques of directed mutagenesis, are GMOs as well and are fully regulated by Directive 2001/18/EC. However, Directive 2001/18/EC only stipulates rules for the deliberate release and placing on the market of GMOs. By contrast, the European Union (EU) has adopted a separate set of rules laid down in Directive 2009/41/EC, which apply to the so-called ‘contained use’ of ‘genetically modified micro-organisms’ (GMMs). Whether also genome-edited micro-organisms are GMMs and, thus, subject to Directive 2009/41/EC is of crucial importance since contained use activities with genome-edited micro-organisms are currently carried out extensively, eg in laboratories and research facilities. An in-depth legal analysis shows that the CJEU’s interpretation of Directive 2001/18/EC can be extended to Directive 2009/41/EC which means that, in the end, genome-edited micro-organisms are GMMs invariably subject to Directive 2009/41/EC.
https://academic.oup.com/jlb/article/9/1/lsab033/6513430
Jany Kl.-D:: Werden genom-editierte Pflanzen anders eingeordnet als genom-editierte Mikroorganismen?
https://www.biotech-gm-food.com/kommentare/eugh-urteil-mutagenese-geschlossene-systeme-rl-2009-49-EG
07.11.2024