Ferreira S., Vauvy G.
GENOSCREEN, Lille, France 

The quality control of probiotic in foods traditionally has relied solely on tests to ensure that an adequate number of viable bacteria are present in the products throughout their shelf lives. Viability is an important factor, but not the only criterion for quality assurance. The literature has already shown that many commercial probiotic preparations lack necessary quality control, as often the probiotic preparations are not only non-viable but also incorrectly identified or, worse, contain microorganisms not recognized as probiotics. Thus, there is a need for strain-specific methodologies to assure consumers of product quality. We here report the development of a method for the strain-specific identification and quantification of five different probiotics mixed in a unique marketed formulation.

The five strains of probiotics (L. casei, L. acidophilus, L rhamnosus GG, B. lactis and S boulardii) constituting the marketed formulation were obtained separately from suppliers as well as the commercial capsules with their exact probiotic composition. Five strain-specific designs were carried out to detect and quantify the strains of interest through a qPCR method. Using an internal optimized protocol, gDNA was isolated from each lyophilized probiotic, from the commercial caps and from an artificial mix made according to the exact composition of the commercial caps. Each separate strain-specific DNA was then used to verify the proper PCR amplification, build and validate each calibration ranges and ensure the absence of cross-reactivity of the qPCR between strains.

Four assays distinguished its specific strain from the other bacteria regardless of whether the DNA was isolated from pure lyophilized strain, the artificial mix or the commercial caps. The calibration curves for each four strain could be established and therefore used for the quantitative control of the strains present in the commercial formulation. On the contrary, the qPCR assay specific for L. acidophilus strain was not effective at all, even on the pure gDNA extracted from the lyophilized form of the probiotic. A second qPCR assay was then designed on another genomic region and tested. Similarly, no detection was obtained for this specific probiotic strain. Two unsuccessful strain-specific qPCR designs being unexpected, we performed two additional molecular controls: the Sanger sequencing of the qPCR targeted region and 16S gene to further confirm strain identity. Surprisingly, we discovered that the qPCR targeted region was much mutated (~19%) compared to an L.acidophilus reference. Comparison of this qPCR specific region and 16S gene sequences to public database highlighted that the strain identified as L. acidophilus is actually a L. johnsonii.

This study further confirm the efficiency of strain-specific molecular methods in the quality control of marketed formulation of mixtures of probiotics as it allows strain-specific identification, detection and quantification but also detection of misspelled bacteria.

Keywords: Molecular Biology, Quality control, Probiotic quantification, Probiotic identification, qPCR

Ferreira S. & Vauvy G. (2016). Development of a molecular quality control for a commercial probiotic formula. Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 91.). IPC2016

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