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Enumeration of Probiotic Strain Lacticaseibacillus rhamnosus GG (ATCC 53103) Using Viability Real-time PCR

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Abstract

Probiotic health benefits are strain specific and are dose dependent. Hence, administering the correct strains, at the recommended doses is essential to achieve probiotic health benefits. Reliable methods are needed to facilitate probiotic strain identification and enumeration. Plate count methods are the most commonly used methods for probiotic enumeration. However, these methods are time-consuming, laborious, highly variable, and non-specific. Here, we developed a real-time PCR method for enumeration of a commonly used strain, Lacticaseibacillus rhamnosus GG. The method utilizes PMAxx as a viability dye to enumerate viable cells only. Optimization of viability treatment showed that PMAxx at a final concentration of 50 μM was effective in inactivating DNA from dead cells, and that bead beating for 5 min at 3000 rpm was effective in liberating DNA. The assay demonstrated high efficiency between 93 and 102%, with R2 values > 0.99. The assay showed high precision with relative standard deviation (RSD%) below 2.3%. Assay performance was compared to a plate count method in which there was a strong correlation between both methods (Pearson r = 0.8443). This method offers a 10 × shorter time for results and a higher precision compared to plate count methods. Furthermore, this method enables specific enumeration of L. rhamnosus GG in multi-strain products, which is not possible to achieve using plate count methods. This novel method facilitates faster and more accurate enumeration of L. rhamnosus GG as a raw ingredient as well as in finished products which enables better quality assurance and efficacy of probiotics for consumers.

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Acknowledgements

The authors thank UAS Labs, International Flavors & Fragrances Inc., Nature’s Way Brands, and Lallemand Health Solutions for kindly providing the reference materials.

Funding

This study was supported by the Natural Health Product Research Alliance (NHPRA), University of Guelph.

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Authors and Affiliations

  1. College of Biological Science, NHP Research Alliance, University of Guelph, Guelph, ON, N1G 2W1, Canada

    Hanan R. Shehata & Steven G. Newmaster

  2. Department of Microbiology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt

    Hanan R. Shehata

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  1. Hanan R. Shehata
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HS designed the study, carried out the experiments, analyzed the data, and wrote the manuscript. SN helped to design the study, facilitated sample acquisition, and edited the manuscript. All the authors read and approved the manuscript.

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Correspondence to Hanan R. Shehata.

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Shehata, H.R., Newmaster, S.G. Enumeration of Probiotic Strain Lacticaseibacillus rhamnosus GG (ATCC 53103) Using Viability Real-time PCR. Probiotics & Antimicro. Prot. 13, 1611–1620 (2021). https://doi.org/10.1007/s12602-021-09849-6

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