Obtaining regulatory approval to use microorganisms as food additive or production strain can be challenging. Applying the regulatory guidelines and rules to the genomic data generated when analysing these microbial strains often presents both technical and interpretation challenges. This article outlines the regulatory requirements and challenges and shows how BaseClear can facilitate to the characterisation of microbial strains for EFSA and FDA file submission.

Common issues with microbial genome sequence data and its interpretation

A number of technical challenges is encountered when the complete genome sequence of a selected microorganism is being characterised. These include problems closing sequence gaps in the genomes, specifically in larger fungal genomes. Additionally, it is often particularly difficult to extract and purify genomic DNA and plasmid DNA of sufficient quality to allow long-read sequencing. This is of particular relevance when sequencing fungal genomes using the PacBio and Oxford Nanopore sequencing platforms. The presence of plasmids in a strain and the correct sequencing and assembly of such novel plasmids can be particularly challenging, as in many cases there are multiple plasmids present which have homologous (or very similar) genes and genetic elements.

Furthermore, in cases where a potential trait of concern is found, such as the presence of a plasmid or a partial antimicrobial gene, the interpretation of this information is critical to the final outcome. Are these traits a safety problem for the use of the microorganism as a food additive or production strain or are they only minor details that have no consequences for safety? And how will this be regarded by the regulatory authorities? In order to address these issues, it is important to have a clear understanding of the current rules and guidelines from the regulatory bodies.

Current guidelines for food additives, production strains and probiotics: The EFSA & FDA requirements

All microorganisms used as food additives or production strains for food additives need to be approved by the appropriate regulatory body, usually either the European Food Safety Authority (EFSA) or the U.S. Food and Drug Administration (FDA). Approval is given if the microorganisms are shown to be safe. However, when applying the regulatory guidelines and rules to the genomic data that is generated when analysing the microbial strains, one is often faced with a number of both technical and interpretation challenges.

In this article we give an overview of these regulatory requirements and the challenges many are facing, and summarise the differences between the EFSA and FDA approaches to this subject. We will also highlight the essential role whole genome sequencing now plays in this process and how BaseClear can help with the required genomics characterisation. BaseClear offers a number of services focused on issues related to the characterisation of microbial strains for EFSA and FDA file submission.

EFSA and FDA differences: QPS vs. GRAS

The EFSA and FDA have different approaches when asked to approve a microorganism for food production or food use. Consequently, their regulatory procedures for approval of applications are also different. They use different starting points, resulting in quite different procedures.

The EFSA has developed a specific Qualified Presumption of Safety (QPS) procedure for the safety risk assessment of biological agents [1]. This procedure only applies to microorganisms. Applicants can request approval of a microorganism, by submitting a technical dossier with extensive information about the microorganism according to the guidance documents provided by the EFSA. When the approval is granted, the applicant is allowed to use this strain as a food additive or production organism. Additionally, when new applications of the microorganism are developed, it is not necessary to request approval again. The focus of the procedure is on the microorganisms and not on the product for which they are used.

The FDA approach is to determine whether the microorganism can be considered GRAS, where GRAS is an acronym for Generally Regarded As Safe [2]. This classification not only applies to microorganisms, but to all types of food additives. In contrast to the EFSA procedure, the focus is on the final product in which the food additive is incorporated. This means that for each new product in which the same additive is used, a new procedure for approval needs to be initiated.

An overview of the most important differences between the GRAS and QPS procedures is given in the table below.

Applies to food additives including microorganisms Applies to microorganisms only
Performed after a specific GRAS notification to FDA Performed for microorganisms used as a source of or contained in products assessed for EU market authorisation
Determination of GRAS status by FDA and/or external experts Determination of QPS status by EFSA
Applicants request a GRAS status EFSA requests evaluation of new taxonomic units upon receiving an application dossier by the EC
Based on history of use, common knowledge (including scientific data) Based on history of use, body of knowledge and  absence of adverse effects
Describes specific substance or microorganism at strain level Describes taxonomic unit (usually species level for bacteria and yeasts, families for viruses, not at strain level)
Case-by-case safety assessment at strain level General safety assessment at species of family level
Based on specific guidance Support to the safety assessment required in the founding EU regulation
Open tool to all applicants Internal tool only under the frame of dossiers for authorisation of regulated products by EFSA

Based on: L. Herman, Role of the Qualified Presumption of Safety concept in the EFSA risk evaluations, presentation in Copenhagen, 2018.

EFSA – QPS requirements

The EFSA’s regulatory framework for characterisation and approval of microorganisms is based on an approved list of organisms that they have characterised as safe (Qualified Presumption of Safety; or QPS). If a microorganism is to be approved and added to the list, the microorganism needs to be thoroughly characterised. The requirements for this characterisation for a QPS procedure depend on the type of food additive that is considered, as shown in the table below [1].

  Food additives containing viable microorganisms Food additives produced by production organisms
  Bacteria Fungi-yeast Bacteria Fungi-Yeast
Identification x x x x
Antimicrobial Susceptibility x x
Antimicrobial production x x x x
Toxigenicity and pathogenicity x x x x
Genetic modification For GMMs only For GMMs only
Absence of the production strain x x
Presence of DNA from the production strain Where relevant Where relevant
Compatibility with other authorised additives Where relevant Where relevant

Taken from [1].

Whole genome sequencing

Whole genome sequencing plays an important role in many of the requirements mentioned above. First of all, whole genome sequencing (WGS) provides the information to unequivocally taxonomically identify the strain that is used. EFSA requires WGS for bacteria and yeasts; for filamentous fungi this is also recommended. Only when WGS is not available for filamentous fungi can one do the identification by comparing 18S rRNA genes, ITS regions and other characteristic genes.

Moreover, WGS has the advantage that it also provides information which enables further characterisation of the strains, specifically regarding potential functional traits, such as:

  • Antimicrobial susceptibility. Microbial food additives should not add to the pool of antimicrobial resistance (AMR) genes that are already present in the gut bacterial population or otherwise increase the spread of antimicrobial resistance. This can be checked by searching the genome for the presence of known AMR genes.
  • The production of antimicrobials. The microorganisms should not produce antimicrobials that are clinically relevant. This can be checked by searching the genome for the presence of known antimicrobial genes.
  • Toxigenicity and pathogenicity. The microorganisms should not cause diseases or produce toxic components. The genome can be searched for example for the presence of genes involved in the production of known toxic metabolites and the presence of virulence factors.
  • Genetic modifications. This only applies to genetically modified production strains. For such strains, the genome can be checked for the introduced modifications.
  • Mobile elements. The genome can be checked for the presence of mobile elements such as plasmids.

For several of these functional traits, additional tests are required to fully prove the safety of the microorganism [1], but whole genome sequencing can help to minimise the efforts on this.

BaseClear offers a complete service for whole genome sequencing. BaseClear has a lot of experience and expertise in whole genome sequencing and microbial strain characterisation. We can do the whole genome sequencing itself as well as further characterisation, such as an antibiotic resistance gene screening, plasmid detection and identification and production strain analysis, including copy number and gene expression analysis.

Reports suitable for EFSA application

BaseClear now offers a new service to help you with the interpretation of such data, based on an additional literature research. Our consultant will judge all data in light of the current knowledge and prepare a report that is suitable for an EFSA application. Contact us if you would like to know more about this service.


  1. EFSA: Guidance on the characterisation of microorganisms used as feed additives or as production organisms, http://www.efsa.europa.eu/en/efsajournal/pub/5206
  2. FDA: Ingredients, Additives, GRAS & Packaging Guidance Documents & Regulatory Information, https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/IngredientsAdditivesGRASPackaging/default.htm

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