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In November 2025, EFSA published a major update to how microorganisms used in the food chain must be characterised. For companies working with fermentation, microbially produced alternative proteins, biomasses, probiotics, microbial enzymes, or microbial production hosts, the new document brings both simplification and new expectations. The guidance replaces the sector‑specific documents that applicants have followed for years, and, crucially, brings all products prepared from, obtained from, or produced with microorganisms under one umbrella.
In our first webinar of 2026, Pauliina Halimaa, Managing Director of Biosafe, walked through the guidance and highlighted the practical consequences for developers preparing EU dossiers. As she noted during the session: “This is now one guidance for all microorganisms used in the food chain: practical for applicants, but with important new expectations.”
One guidance, new expectations
The idea behind the new document is straightforward. Instead of navigating different microbial requirements across novel foods, feed additives, food enzymes, and GM applications, developers can now rely on a single set of principles. This consolidation also means that certain requirements have been tightened or clarified.
A new key concept is EFSA’s categorisation of microbial products into active agents, biomasses and production strains. These categories are not legal definitions, but practical tools for aligning data requirements. Active agents are microorganisms capable of multiplication and now formally include bacteriophages, with their host classified as the production strain. Biomasses consist of inactivated cells. Production strains are microorganisms that produce substances but are removed from the final product.
Another early clarification concerns QPS status, which can now also apply to genetically modified microorganisms as long as no genes of concern remain. If a gene of concern is removed by genetic modification, that may fulfil the qualification. This nuance is important: a microorganism may be QPS from a safety assessment perspective yet still fall under GMO regulations depending on the product and process.
Taxonomy, WGS and the two‑year rule
One of the most significant updates is the strengthened requirement for taxonomic identification at species level. Whole genome sequencing is now mandatory for bacteria, yeasts, filamentous fungi and viruses. Microalgae and protists may be identified using morphological and marker‑gene data, though WGS remains advantageous because it supports other parts of the assessment.
EFSA also introduces a freshness rule: bioinformatic analyses may not be older than two years at the time of application. As Pauliina explained, this typically means re-running the database screenings rather than re-sequencing the strain, assuming the organism itself has not changed.
AMR: still the most debated section
Antimicrobial resistance continues to be one of the most challenging components of microbial risk assessment. EFSA now requires screening against all antimicrobials of medical or veterinary relevance, broadening the scope especially for feed applications. The decision tree introduced in the guidance places heavy weight on whether an AMR gene is acquired or intrinsic, and whether horizontal gene transfer (HGT) is plausible.
Determining intrinsicity is not simple. Applicants must examine all non‑clonal strains of the species, ideally at least 30 of them. EFSA has not yet defined how many SNPs constitute clonality, which leaves room for interpretation and discussion. As Pauliina noted, this strict approach could make it difficult for new or poorly represented species to enter the market if genomic data is lacking.
If uncertainty remains, EFSA considers the gene acquired, even when evidence might lean toward intrinsicity. This conservative stance is likely to shape dossier strategies for years to come.
For bacteria, phenotypic testing is required when a gene is acquired or its nature is uncertain. However, EFSA now expects applicants to focus only on the relevant antimicrobials rather than broad panels. Yeasts and filamentous fungi, when used as active agents, must undergo antifungal susceptibility testing. Not for HGT concerns, but to ensure that infections in humans or animals would remain treatable.
Do microorganisms produce antimicrobial compounds?
Another notable section concerns the production of antimicrobial substances. EFSA requires both WGS‑based evaluation of biosynthetic clusters and phenotypic confirmation when the species is non‑QPS or otherwise known to produce relevant metabolites. If gene clusters associated with therapeutic antimicrobials are detected, applicants must quantitatively analyse culture supernatants or final products. Even trace amounts may raise concerns due to the risk of sub‑inhibitory exposure.
Microalgae used as feed additives form the main exception, following FEEDAP’s conclusion that they are not expected to produce clinically relevant antimicrobials.
GMMs
For genetically modified strains, EFSA still requires accurate and thorough description of all modifications. Applicants must describe inserted sequences, donor organism taxonomy, designed elements, deletions, substitutions, amino acid sequences, function, and mapping of all functional components. The comparison to the parental strain should be WGS‑based, and EFSA increasingly emphasises documenting the parental strain’s origin and modification history, something that can be difficult for older or heavily optimised strains.
What EFSA expects from final product testing
EFSA has clarified requirements for showing the absence of viable cells or DNA in fermentation products and biomasses. Viability must now be assessed on solid media, using prolonged incubation and conditions that favour the recovery of stressed cells. Positive controls, where the production strain must grow in the presence of the product, can be technically demanding, especially when the product matrix inhibits growth.
For DNA, EFSA recommends a semi‑quantitative PCR approach. The commonly cited 10 ng/g threshold is not an allowed level in the product; it is the limit of detection the method must achieve. If DNA from a strain carrying a gene of concern is detected at any level, it is considered a risk.
Environmental risk assessment in context
Environmental assessments apply to active agents and biomasses, focusing on primary and secondary exposure routes. QPS strains are generally not environmental risks. Non‑QPS strains that already occur in the receiving microbiome may also be acceptable. Biomasses without genes of concern usually pass easily. The more complex ERA requirements concern genetically modified active agents, still largely theoretical, as no such applications are currently under EFSA review.
Questions from the audience
The webinar ended with an active Q&A session, reflecting how many companies are already preparing dossiers under the new framework. Below are a few highlights that capture common worries and areas where EFSA’s guidance leaves room for interpretation.
Q: Does the two‑year rule mean I must re‑sequence my strain?
A: Typically no. Only the bioinformatic analyses must be updated, assuming the original sequencing meets current quality standards and the strain has not been further modified.
Q: Does AMR assessment apply to fungi and yeasts?
A: No. These require antifungal susceptibility testing rather than AMR gene evaluation.
Q: How do I plate 1 g of dense biomass for viable‑cell testing?
A: By using multiple plates and suitable dilutions. Tailored methods are often necessary.
Q: Should PCR targets be strain‑specific?
A: It is no longer mandatory, but it is still recommended to avoid detecting contaminants.
Q: When can an AMR gene be considered intrinsic?
A: EFSA provides no strict threshold. High prevalence among non‑clonal strains supports intrinsicity, while borderline cases remain uncertain.
From guidance to submission: how Biosafe helps
As Pauliina emphasised at the end of the webinar, microbial characterisation is only one piece of an EU application, but it is one of the most technically intensive parts. Our team combines regulatory expertise with microbiology, bioinformatics, molecular biology and wet‑lab capability. We routinely support applicants with AMR intrinsicity analyses, phenotypic testing, viable cell testing from challenging matrices, strain‑specific PCR design, and dossier preparation.
If you're planning a submission in 2026, now is a good time to align your strategy with the new guidance.
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Read more: Microbial testing – An overview of what it is and what it is for
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