Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage

Abstract

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Probiotics are typically enumerated by agar plate counting (PC) techniques. PC has several limitations including poor specificity, high variability, inability to enumerate dead cells, viable but non-culturable cells and cells in complex matrices. Viability droplet digital polymerase chain reaction (v-ddPCR) is an emerging enumeration technique with improved specificity, precision, and the ability to enumerate cells in varying states of culturability or in complex matrices. Good correlation and agreement between v-ddPCR and PC is well documented, but not much research has been published on the comparison when enumerating freeze-dried (FD) probiotics during storage. In this study, v-ddPCR utilizing PE51 (PE51-ddPCR), a combination of propidium monoazide (PMA) and ethidium monoazide (EMA), was evaluated as alternative enumeration technique to PC on blends of four FD probiotic strains over the course of a 3-month storage study with accelerated conditions. When PMA and EMA are combined (PE51), this study demonstrates agreement (bias = 7.63e+9, LOA = 4.38e+10 to 5.9e+10) and association (r = 0.762) between PC and v-ddPCR, at or above levels of an accepted alternative method. Additionally, v-ddPCR with individual dyes PMA and EMA provide insight into how they individually contribute to the viable counts obtained by PE51-ddPCR and provide a more specific physiological understanding of how probiotics cope with or experience damage during storage.

Keywords

• probiotic
• storage
• v-ddPCR
• viability
• enumeration
• PMA