Quantification of pasteurized Akkermansia muciniphila MucT by flow cytometry: protocol optimization and inter-laboratory ring test

Akkermansia muciniphila Muc T was authorized by EFSA in 2021 to be commercialized as novel food in its pasteurized form, corresponding to what it is commonly referred to as a postbiotic. As for probiotics, which are commercialized as living cells, postbiotics also require their correct quantification during the production process. Here we describe the optimization of a flow cytometry-based protocol for the absolute counting of pasteurized A. muciniphila cells in high cell density and standardized industrial batches. Protocol optimization required a sample preparation set-up and the choice of the appropriate diluent for the efficient rehydration of freeze-dried cells to limit cell aggregates. The developed quantification protocol allowed a more efficient cell counting with a lower standard deviation and coefficient of variation (CV) as compared with microscope-based quantification. The optimized protocol was successfully applied in a mass balance calculation over the production process, a standard validation in every industrial process for microbial biomass production that starts from high cell density batches to achieve standardized batches with the target cell dosage. Finally, the inter-laboratory trial, carried out among six independent laboratories using a ring test design, revealed good accuracy and precision of the optimized quantification protocol with an average CV ranging between 12.3 and 24.1 % and a Z score max of 2.64. In conclusion, our study revealed that developed flow cytometry protocol is a fast, reliable, reproducible and accurate method for the enumeration of the pasteurized A. muciniphila Muc T that could be applied with minor modifications to other postbiotics. • Optimization of a flow cytometry protocol for pasteurized A. muciniphila Muc T • Sample preparation and diluent selection minimized cell aggregation • Protocol is suitable for mass balance validation in industrial application • Inter-laboratory ring test confirmed accuracy and precision of the protocol