New Insights into Probiotic Enumeration in a Complex Food Matrix: Flow Cytometry and Plate Count Correlation

Accurate probiotic enumeration ensures the dose delivered to the mammalian host. Plate count (PC) detects only culturable cells and can underestimate the total number of viable cells, particularly when some cells are injured or dormant and do not form colonies.

To qualify a flow cytometry assay reporting active fluorescent units (AFU) and to compare counts with the PC method for micro-encapsulated Lacticaseibacillus rhamnosus GG in a snack product.

Following AOAC INTERNATIONAL, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), and United States Pharmacopeia (USP) principles, precision, accuracy, ruggedness, specificity, and robustness of plate count and flow cytometry methods were evaluated for the first time in a commercial yogurt-bite snack product.

Both methods fulfilled the pre-specified performance targets (RSD ≤15%; AFU recovery 100-104%; live/dead R2 ≥0.95). Analyst-to-analyst differences were not significant. Flow cytometry spike recoveries were 100-104% across targets. Live/dead mixtures tracked linearly (R2 ≥0.95). Across matched samples, AFU and CFU values were equivalent within a pre-specified ±0.5 log band; batch means typically differed by ≤0.2 log with no systematic bias.

The qualified flow cytometry method provides same-day counts and enumerates all membrane-intact cells, complementing plate counts while meeting analytical performance criteria. Findings endorse the use of AFU in tandem to CFU assessment for probiotic quantification, label accuracy and estimation of microbiome-relevant dose in snack and finished-product matrixes.

Flow cytometry provided same-day results and enumerated all membrane-intact cells for label verification in snack matrixes.