Intercompany Validation of Probiotic Enumeration Methods
Interlaboratory validation of culture-based and flow cytometry methods for quantifying microencapsulated probiotics in complex food matrices
Andrzej Benkowski1, Eric Williams1
Gonzalo Saiz-Gonzalo2, Orlaith O’Connell2, Shima Joy2, Seasum McSweeney2, and Sinead B. Bleiel2
1. Eurofins Microbiology Laboratories, Inc, Madison, WI, USA
2. AnaBio Technologies, Carringtwohill, Cor, T455RW24, Ireland
This intercompany study demonstrates the equivalence of plate count (CFU) and flow cytometry (AFU) methods for enumerating microencapsulated Lacticaseibacillus rhamnosus GG in a yogurt-bite matrix. Both methods met validation criteria with strong precision (RSD ≤15%) and agreement within ±0.5 log. Flow cytometry additionally enabled rapid, same-day results with high accuracy and specificity. These findings support a dual-method strategy to enhance speed, reliability, and confidence in probiotic label claims and quality control.
Objective
To conduct an interlaboratory evaluation and validation of complementary enumeration methods for microencapsulated Lacticaseibacillus rhamnosus GG in a yogurt-bite matrix and to assess equivalence between plate count (CFU) and flow cytometry (AFU) approaches for probiotic quantification.
Methods
Both methods met the validation criteria, demonstrating acceptable precision (RSD ≤15%) and no statistically significant differences across operators, days, and experimental conditions. Across all datasets, AFU and CFU values were equivalent within ±0.5 log, supporting analytical comparability. Flow cytometry additionally demonstrated high accuracy (100-104% recovery) and specificity (R2 ≥0.95), while enabling same-day results.
Results
Both methods met the validation criteria, demonstrating acceptable precision (RSD ≤15%) and no statistically significant differences across operators, days, and experimental conditions. Across all datasets, AFU and CFU values were equivalent within ±0.5 log, supporting analytical comparability. Flow cytometry additionally demonstrated high accuracy (100-104% recovery) and specificity (R2 ≥0.95), while enabling same-day results.
Conclusions
This interlaboratory study demonstrates that both plate count and flow cytometry methods are robust, reproducible, and demonstrate equivalence for enumeration of microencapsulated probiotics in complex food matrices. The strong agreement across laboratories supports the use of a dual-method approach, where flow cytometry provides rapid, actionable results and plate count serves as the regulatory reference, enhancing confidence in probiotic label claims and quality control.
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