Assessing viability and stress tolerance in multi-species probiotic products

Probiotic products are expected to contain abundant populations of viable microorganisms that can tolerate environmental stressors. Industry standards for determining viability rely on the plate counting method. However, this cannot enumerate metabolically active yet unculturable bacteria. Therefore, this study developed flow cytometry-based protocols for enumeration of probiotic bacteria and compared plate counting and flow cytometry techniques for their ability to determine the impact of storage temperatures and gastrointestinal digestion on microbial cell viability of multi-species probiotic product IBS Relief®.

Flow cytometry protocols were optimized using Lactobacillus rhamnosus isolated from IBS Relief®. Product viable cell counts were determined using both techniques, utilizing SYTOXTM Green staining to distinguish membrane compromised and intact cells. Flow cytometry routinely resulted in significantly higher intact cell counts than plate counting. Flow cytometry assays showed low temperatures had minimal impacts on cell survivability, while exposure to 37ºC and 50ºC led to 28% and 67% reduction of intact cells, respectively. Similarly, conditions mimicking gastrointestinal digestion (pepsin and bile salts assays), led to 54% and 43% reduction of intact cells, respectively. This work demonstrates that flow cytometry is suitable for rapid and accurate enumeration of viable cells in mixed-species probiotics and has the potential to replace plate counting.