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Assessing probiotic viability in mixed species yogurt using a novel propidium monoazide (PMAxx)-quantitative PCR method.

ABSTRACT: Viability is a prerequisite for any therapeutic benefits associated with the ingestion of probiotic bacteria. Current culture-based techniques are inadequate for the enumeration of probiotics in mixed-species food products. This study utilized a quantitative PCR (qPCR) method coupled with propidium monoazide (PMAxx), and novel species-specific tuf gene primers to selectively enumerate Lacticaseibacillus rhamnosus, Bifidobacterium spp., and yogurt starter cultures in mixed-species probiotic yogurt. The method was optimized for PMAxx concentration and specificity and evaluated for efficiency and applicability. PMAxx-qPCR showed high specificity to the target organisms in mixed-species yogurt, quantifying only viable cells. The linear dynamic ranges were established over five to seven orders of magnitude. The assay was reliable with an efficiency of 91-99%, R² values > 0.99, and a good correlation to the plate count method (r = 0.882). The results of this study demonstrate the high selectivity, improved lead time, and reliability of PMAxx-qPCR over the culture-dependent method, making it a valuable tool for inline viability verification during processing and improving probiotic quality assurance for processors and consumers.

SUBMITTER: Marole TA

PROVIDER: S-EPMC10882272 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature

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Assessing probiotic viability in mixed species yogurt using a novel propidium monoazide (PMAxx)-quantitative PCR method.

Marole Tlaleo A TA Sibanda Thulani T Buys Elna M EM

Frontiers in microbiology 20240208

Viability is a prerequisite for any therapeutic benefits associated with the ingestion of probiotic bacteria. Current culture-based techniques are inadequate for the enumeration of probiotics in mixed-species food products. This study utilized a quantitative PCR (qPCR) method coupled with propidium monoazide (PMAxx), and novel species-specific <i>tuf</i> gene primers to selectively enumerate <i>Lacticaseibacillus rhamnosus, Bifidobacterium</i> spp., and yogurt starter cultures in mixed-species p ...[more]

PMID: 38389538

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Project description:BackgroundHigh-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g., qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting "viable" communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark "PMA-seq" (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities.ResultsPMA-seq was able to successfully reconstruct simple synthetic communities comprising viable/heat-killed Escherichia coli and Streptococcus sanguinis. However, in realistically complex communities (computer screens, computer mice, soil, and human saliva) with E. coli spike-in controls, PMA-seq did not accurately quantify viability (even relative to variability in amplicon sequencing), with its performance largely affected by community properties such as initial biomass, sample types, and compositional diversity. We then applied this technique to environmental swabs from the Boston subway system. Several taxa differed significantly after PMA treatment, while not all microorganisms responded consistently. To elucidate the "PMA-responsive" microbes, we compared our results with previous PMA-based studies and found that PMA responsiveness varied widely when microbes were sourced from different ecosystems but were reproducible within similar environments across studies.ConclusionsThis study provides a comprehensive evaluation of PMA-seq exploring its quantitative potential in synthetic and complex microbial communities, where the technique was effective for semi-quantitative purposes in simple synthetic communities but provided only qualitative assessments in realistically complex community samples. Video abstract.

Dataset Information

Assessing probiotic viability in mixed species yogurt using a novel propidium monoazide (PMAxx)-quantitative PCR method.

Publications

Assessing probiotic viability in mixed species yogurt using a novel propidium monoazide (PMAxx)-quantitative PCR method.

Similar Datasets

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