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

Area of Science:

• Microbiology
• Analytical Chemistry
• Food Science

Background:

• Accurate probiotic enumeration is crucial for determining the delivered dose to the host.
• Plate counting (PC) only detects culturable cells and may underestimate viable cells, especially if injured or dormant.

Purpose of the Study:

• To validate a flow cytometry assay for quantifying active fluorescent units (AFU) of probiotics.
• To compare AFU counts with traditional plate counts (PC) for micro-encapsulated Lacticaseibacillus rhamnosus GG in a snack product.

Main Methods:

• Evaluation of plate count and flow cytometry methods based on AOAC/ICH/USP principles.
• Assessment of precision, accuracy, ruggedness, specificity, and robustness in a commercial snack product.

Main Results:

• Both methods met performance targets (%RSD ≤ 15; AFU recovery 100-104%; live/dead R2 ≥ 0.95).
• Flow cytometry provided same-day results with high spike recovery (100-104%) and linear live/dead tracking (R2 ≥ 0.95).
• AFU and colony-forming unit (CFU) counts were equivalent within ±0.5 log, with minimal batch mean differences (≤0.2 log) and no systematic bias.

Conclusions:

• The validated flow cytometry method provides rapid, same-day enumeration of membrane-intact probiotic cells.
• This method complements PC by quantifying all viable cells, aiding label accuracy and dose estimation in food products.
• Findings support using AFU alongside CFU for comprehensive probiotic quantification in snack matrices.