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Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage
Anthony Kiefer1, Phillip M Byrd1, Peipei Tang1
1IFF Health and Biosciences, Danisco USA, Inc., Madison, WI, United States.
Abstract:
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.
Insights
Viability droplet digital PCR (v-ddPCR) offers a more accurate method for enumerating freeze-dried probiotics compared to traditional plate counting. This advanced technique provides better specificity and precision, especially for probiotics stored under stress conditions.
Area of Science:
- Microbiology
- Molecular Biology
- Food Science
Background:
- Traditional plate counting (PC) for probiotics has limitations in specificity, variability, and enumeration of non-culturable or dead cells.
- Viability droplet digital PCR (v-ddPCR) is an emerging technique offering improved precision and specificity for cell enumeration, even in complex matrices.
- Limited research exists on comparing v-ddPCR with PC for enumerating freeze-dried (FD) probiotics during storage.
Purpose of the Study:
- To evaluate v-ddPCR using a combination of propidium monoazide (PMA) and ethidium monoazide (EMA), termed PE51-ddPCR, as an alternative enumeration method for FD probiotics.
- To assess the performance of PE51-ddPCR against PC over a 3-month accelerated storage study.
- To investigate the individual contributions of PMA and EMA in v-ddPCR for understanding probiotic viability during storage.
Main Methods:
- Utilized PE51-ddPCR, combining PMA and EMA, for enumerating blends of four FD probiotic strains.
- Conducted a 3-month storage study under accelerated conditions.
- Compared enumeration results from PE51-ddPCR with traditional PC.
Main Results:
- Demonstrated agreement (bias = 7.63e+9, LOA = 4.38e+10 to 5.9e+10) and association (r = 0.762) between PE51-ddPCR and PC, meeting criteria for an accepted alternative method.
- PE51-ddPCR showed performance at or above the levels of accepted alternative methods.
- Individual dye analysis (PMA and EMA) provided insights into probiotic physiological states during storage.
Conclusions:
- PE51-ddPCR is a viable and accurate alternative to PC for enumerating FD probiotics during storage.
- v-ddPCR with individual dyes offers a more specific physiological understanding of probiotic responses to storage stress.
- This method enhances the reliability of probiotic enumeration, particularly for products undergoing storage challenges.
