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

Akkermansia muciniphila MucT 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 MucT that could be applied with minor modifications to other postbiotics.