Simultaneous Enumeration of Spore-Forming Probiotics and Postbiotics in Multi-Strain Blends Using Flow Cytometry : Adaptation of ISO 19344 Protocol B
Simultaneous Enumeration of Spore-Forming Probiotics and Postbiotics in Multi-Strain Blends Using Flow Cytometry
Andrzej Benkowski, Eric Williams, Katelyn Sesolak, Mollie Comella -Eurofins Microbiology Laboratories, Inc, Madison, WI, USA
Postbiotic and spore-forming probiotics are among the most popular supplement products on the market. However, there is a notable gap in current testing methodologies, as no standardized approach exists for accurately enumerating both components simultaneously in blended products.
Objective
To develop and validate a single-panel flow cytometry method for simultaneous enumeration of spore-forming probiotics and postbiotics in multi-strain blends, adapted from ISO 19344 Protocol B.
Methods
Six strains were evaluated individually using ISO 19344 Protocol B-adapted flow cytometry with added sonication to disrupt aggregates: three spore-forming probiotics (Heyndrickxia coagulans) (f.k.a. Bacillus coagulans), Shouchella clausii (f.k.a. Bacillus clausii), Bacillus subtilis complex) and three heat-inactivated postbiotics (Lactococcus lactis, Akkermansia muciniphila, Lactobacillus crispatus). Enumeration was then performed on three blended formulations: a multi-strain spore blend, a multi-strain postbiotic blend, and a combined spore–postbiotic blend. Gating strategies based on scatter and fluorescence characteristics were developed to resolve spore and postbiotic populations within each blend.
Results
For individual spore strains, flow cytometry showed strong agreement with standard plate counts for all probiotic spore-formers, with log differences of +0.16 (H. coagulans), −0.18 (S. clausii), and +0.10 (B. subtilis). Analysis of blended formulations demonstrated accurate and distinct population recovery relative to theoretical inputs. Measured values closely matched expected counts for the spore-forming probiotic blend (+0.19 log difference), the postbiotic blend (−0.13 log difference), and the combined spore–postbiotic blend (−0.03 log difference for the postbiotic component and +0.16 log difference for the spore component).
Conclusions
This method enables rapid, simultaneous enumeration and differentiation of spore-forming probiotics and postbiotics in complex blends, providing a scalable tool for quality assurance, safety verification, and label claim substantiation.
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