Food Enzyme Database (FEDA): a web application gathering information about food enzyme preparations available on the European market.

Following the European Commission No. 1332/2008 regulation and the consequent necessity of a scientific evaluation of food enzymes (FEs) for their approval for sale on the European Union market, many FE dossiers have been submitted to the European Commission and various documents currently co-exist. In order to centralize all relevant information in one structured location that is easily accessible to support enforcement laboratories and the competent authorities, we developed a web application, called Food Enzyme Database (FEDA). FEDA allows searching and collection of information originating from many different sources in one centralized portal. Queries can be performed using key information types, which include information on the producing company, production source (strain type, genetically modified microorganism status), type of enzyme protein and evaluation status with employed evaluation criteria. The database contains all current publicly available information. Centralizing all information coupled with intuitive searching functionality also allows the generation of general statistics regarding the current market situation. FEDA is open access and is freely available at the following location: https://feda.sciensano.be. Database URL : https://feda.sciensano.be.

Are antimicrobial resistance genes key targets to detect genetically modified microorganisms in fermentation products?

As genetically modified microorganisms (GMM), commonly used by the food and feed industry to produce additives, enzymes and flavourings, are frequently harbouring antimicrobial resistance (AMR) genes as selection markers, health and environmental concerns were consequently raised. For this reason, the interest of the competent authorities to control such microbial fermentation products has strongly increased, especially since several recent accidental contaminations of unauthorized GMM, or associated recombinant DNA, in bacterial fermentation products intended for the European food and feed chain. However, no global screening strategy is currently available in enforcement laboratories to assess the presence of GMM harbouring AMR genes and/or the presence of full-length AMR genes. Moreover, the confidentiality of the related GMM dossiers strongly hampers the development of methods to perform such control. To overcome this issue, an analysis of related publicly available patents was performed in this study to identify all reported AMR genes. On this basis, the aminoglycoside adenyltransferase (aadD) gene, conferring a resistance to both kanamycin and neomycin, was identified as a key target to cover a large spectrum of GM bacteria. A real-time PCR method to screen for its potential presence as well as a nested-PCR method associated with a sequencing analysis to assess its full-length were developed to target this aadD gene. The performance of these new methods were successfully evaluated in terms of specificity, sensitivity and applicability, allowing their easy implementation in enforcement laboratories. Moreover, the integration of these newly developed methods to our very recently proposed strategy, initially targeting GMM carrying a chloramphenicol resistance gene, allows to drastically increase the detection spectrum of GM bacteria producing fermentation food and feed products. The data generated by the proposed strategy represents therefore a crucial support for the competent authorities, especially to evaluate potential risks for the food and feed safety.

Genetically Modified Micro-Organisms for Industrial Food Enzyme Production: An Overview.

The use of food enzymes (FE) by the industrial food industry is continuously increasing. These FE are mainly obtained by microbial fermentation, for which both wild-type (WT) and genetically modified (GM) strains are used. The FE production yield can be increased by optimizing the fermentation process, either by using genetically modified micro-organism (GMM) strains or by producing recombinant enzymes. This review provides a general overview of the different methods used to produce FE preparations and how the use of GMM can increase the production yield. Additionally, information regarding the construction of these GMM strains is provided. Thereafter, an overview of the different European regulations concerning the authorization of FE preparations on the European market and the use of GMM strains is given. Potential issues related to the authorization and control of FE preparations sold on the European market are then identified and illustrated by a case study. This process highlighted the importance for control of FE preparations and the consequent need for appropriate detection methods targeting the presence of GMM, which is used in fermentation products.

Strategy for the identification of micro-organisms producing food and feed products: Bacteria producing food enzymes as study case.

Recent European regulations require safety assessments of food enzymes (FE) before their commercialization. FE are mainly produced by micro-organisms, whose viable strains nor associated DNA can be present in the final products. Currently, no strategy targeting such impurities exists in enforcement laboratories. Therefore, a generic strategy of first line screening was developed to detect and identify, through PCR amplification and sequencing of the 16S-rRNA gene, the potential presence of FE producing bacteria in FE preparations. First, the specificity was verified using all microbial species reported to produce FE. Second, an in-house database, with 16S reference sequences from bacteria producing FE, was constructed for their fast identification through blast analysis. Third, the sensitivity was assessed on a spiked FE preparation. Finally, the applicability was verified using commercial FE preparations. Using straightforward PCR amplifications, Sanger sequencing and blast analysis, the proposed strategy was demonstrated to be convenient for implementation in enforcement laboratories.

Identification of an unauthorized genetically modified bacteria in food enzyme through whole-genome sequencing.

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.