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A novel pharmaceutical approach for the analytical validation of probiotic bacterial count by flow cytometry

Luca Michelutti, Michela Bulfoni, Emanuele Nencioni
Journal of microbiological methods 2020 v.170 pp. 105834
Bifidobacterium, Lactobacillus, acidification, bacteria, biochemical pathways, cell viability, chemical species, computer software, data analysis, dietary supplements, drugs, fermentation, flow cytometry, fluorescent dyes, humans, oxygen, plate count, probiotics, propidium, thiazoles
Introduction: Flow cytometry is a powerful and sensitive technique able to characterize single cells within a heterogeneous population. Different fluorescent dyes can be combined and used together to analyze a great variety of parameters simultaneously. In particular, flow-cytometry allows to measure viability and vitality of probiotics measuring their metabolic activity, fermentation capacity, acidification potential or oxygen uptake ability (Hayouni et al., 2008). To now, plate counting is considered the gold standard in microbiological technique for probiotic enumeration. However, this approach is limited to the detection of only those viable cells which are able to proliferate and form colonies on a solid medium but is not able to recognize not cultivable bacteria and nonviable cells. Aim: The aim of the present study was to apply The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) parameters for the validation of new analytical methods in microbiology. ICH requirements, which are commonly employed for the analysis of drugs and chemical analytes, have been here applied to live cells for the comparison between a flow-cytometric assay and the traditional plate count method for the quantification of viable probiotics bacteria.Methods and results: Combining specific viability dyes such as thiazole orange (TO) and propidium iodide (PI), probiotic counts of Lactobacillus and Bifidobacterium species were carried out using a FACS Verse (BD Biosciences) cytometer. Analyses were conducted in parallel with the traditional plate count, on specific media. Raw data were analyzed using the FACSuite software (BD Biosciences) and then elaborated with the statistical software Neolicy (VWR International). Results indicated that flow cytometry provides very similar results in cell counting if compared to classical microbiology approaches, showing better performances (ICH parameters) than the traditional plate count method.Conclusions: This work demonstrated the analytical ICH validation of probiotic counts in food supplement products using a robust flow cytometric approach able to enumerate and to assess bacteria viability with stronger results in comparison to the traditional plate count.