High resolution analysis of multi-strain probiotic products by NGS using innovative molecular markers
Objective
Analysis of the microbial composition of multi-strains probiotic products is a challenging task. NGS metabarcoding brought great advantages as fast analytical method to understand the microbial communities’ composition for research purposes (1). However, its application in industrial settings for quality control and stability check of probiotics, fermented foods and other products containing microbes, is very limited by the low biodiversity of species used for formulations (2) and by the lack of standard methods that can distinguish strains belonging to same species (3). The newly developed method brings together fast and standardized approach of NGS metabarcoding with an improved accuracy in distinguishing close related species, subspecies and strains of the same species in a blend.
Method
Our method relies on HPME markers (4) which are intergenic highly variable sequences flanked by conserved coding sequence that allows PCR primers design. The amplification, sequencing of HPME markers and matching with a manually curated qualified database, has been first validated on mock communities and then successfully applied for the identification of all species and subspecies in commercial probiotic products.
Results & Conclusions
HPME-based metabarcoding analysis allows to quickly detect and identify species and subspecies in a sample to confirm its composition reported in the label. According to species biodiversity and the presence of known signature sequences, it can also discriminate strains belonging to the same species. Moreover HPME-based metabarcoding can be coupled with standard 16S rRNA metabarcoding to reveal unexpected bacterial species and combined with flow-cytometry to provide relative quantification of each viable strain in the blend.
Acknowledgments
The presented work is a deliverable of the TITAN project funded by the European Union within the Horizon Europe framework (HORIZON-CL6-2021-FARM2FORK-01-17 grant agreement 101060739). https://titanproject.eu/
References
1. Srinivas M, O’Sullivan O, Cotter PD, Sinderen DV, Kenny JG. 2022. The Application of Metagenomics to Study Microbial Communities and Develop Desirable Traits in Fermented Foods. Foods. 11, 3297. doi: 10.3390/foods11203297.
2. Mazzantini D, Calvigioni M, Celandroni F, Lupetti A, Ghelardi E. 2021. Spotlight on the Compositional Quality of Probiotic Formulations Marketed Worldwide. Front Microbiol. 12:693973. doi: 10.3389/fmicb.2021.693973.
3. Dreier M, Meola M, Berthoud H, Shani N, Wechsler D, Junier P. 2022. High-throughput qPCR and 16S rRNA gene amplicon sequencing as complementary methods for the investigation of the cheese microbiota. BMC Microbiol. 22, 48. doi: 10.1186/s12866-022-02451-y.
4. Fracchetti F, Torriani S, Del Casale A, Felis G. 2018. Highly Polymorphic and Modular Extragenic (H.P.M.E.) markers within specific taxa of microorganisms and use thereof for their differentiation, identification and quantification. Patent WIPO (PCT) WO2018015572A1.