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To assess the stability of 16S rRNA of viable but nonculturable (VBNC) probiotics during storage when compared with different attributes of viability.Levels of RNA of the probiotic strains Bifidobacterium longum 46, B. longum 2C and B. animalis subsp. lactis Bb-12 were monitored during storage in fermented and nonfermented foods. Cells which gradually lost their culturability in fermented products retained high level of rRNA, whereas rRNA of acid-killed control cells decreased at faster rate. Furthermore, the viability of B. longum 2C was monitored during storage by measuring changes in reductase activity, cytoplasmic membrane integrity and esterase activity using a flow cytometer. All of the culture-independent viability assays suggested that the cells remained viable during storage. In nonfermented media, the observed losses in culturability were smaller, and the changes in cell counts were comparable with the changes in rRNA levels.Viable but nonculturable probiotics maintain high levels of rRNA and retain properties of viable bacteria including reductase activity. Quantification of 16S rRNA complements culture-independent viability assays.Culture-independent viability assays allow the detection of VBNC probiotics, and can be used parallel to conventional culture-dependent methods to obtain accurate information on probiotic viability.