| Summary: | There is growing scientific and commercial interest in multi-species probiotic products due to their potential benefits in maintaining gut health. Determining the viability of probiotic microorganisms in these products is essential to ensure that they confer maximal health benefits. The gold standard for enumerating probiotic viability is the plate count method. However, this may be inaccurate for enumerating mixed probiotic populations, with recognised limitations including difficulty measuring metabolically active yet unculturable, very slow growing microbes, microencapsulated, enteric coated microbes, or multi-strain formulations that require differing growth media. Here, we developed a flow-cytometry-based approach using SYTOX<sup>TM</sup> Green dye to assess the viability of probiotic microorganisms in a multi-species, fibre-containing probiotic product and compared this to the traditional plate count method. This method was suitable for enumerating both total bacterial cells and the viable cell fraction in the complete product mixture, and could also be used to assess how stressors, such as gastric digestion and exposure to bile acids, affect bacterial cell viability. Flow cytometry measurements routinely detected higher viable cell counts than plate counting. This work demonstrates that flow cytometry assays can be established as a suitable method for rapid enumeration of viable cells in complex, multi-species probiotics.
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