The Probiotic Identity Card: A Novel "Probiogenomics" Approach to Investigate Probiotic Supplements.

Gabriele Andrea Lugli,Giulia Longhi,Francesca Turroni,Chiara Tarracchini,Federico Fontana,Marco Ventura,Giulia Alessandri,Douwe Van Sinderen,Christian Milani,Leonardo Mancabelli,Francesco Di Pierro

doi:10.3389/fmicb.2021.790881

Abstract

Probiotic bacteria are widely administered as dietary supplements and incorporated as active ingredients in a variety of functional foods due to their purported health-promoting features. Currently available probiotic products may have issues with regards to their formulation, such as insufficient levels of viable probiotic bacteria, complete lack of probiotic strains that are stated to be present in the product, and the presence of microbial contaminants. To avoid the distribution of such unsuitable or misleading products, we propose here a novel approach named Probiotic Identity Card (PIC), involving a combination of shotgun metagenomic sequencing and bacterial cell enumeration by flow cytometry. PIC was tested on 12 commercial probiotic supplements revealing several inconsistencies in the formulation of five such products based on their stated microbial composition and viability.

Highlights

In 2009 a novel discipline called probiogenomics was coined to provide insights into the diversity of probiotic bacteria aimed at revealing the molecular basis for their health-promoting activities (Ventura et al, 2009)
Flow cytometry (FC) assays were performed using serial dilutions of the probiotic supplements, in order to enumerate bacterial cells and reveal their viability using dyes capable to distinguish live cells from dead cells based on cell membrane integrity
Another important step of the Probiotic Identity Card (PIC) protocol includes the probiotic genome sequence reconstruction based on Whole Metagenome Shotgun (WMS) data obtained from the sequencing methodology, and the completeness of the assembled chromosomes was further validated using in silico programs aimed at identifying marker genes

Summary

Introduction

In 2009 a novel discipline called probiogenomics was coined to provide insights into the diversity of probiotic bacteria aimed at revealing the molecular basis for their health-promoting activities (Ventura et al, 2009). In this context, the availability of probiotic genome sequences significantly expanded our understanding of the biology of these microorganisms (Ventura et al, 2012). Classical microbiological techniques are currently considered the gold standard for probiotic identification, classification, and enumeration (Chiron et al, 2018) These techniques are time-consuming and not always accurate when it comes to bacterial identification. Next-generation sequencing technologies have enabled accurate evaluation of the relative abundance of (probiotic) microbes in a sample by targeting the 16S rRNA-encoding gene, thereby avoiding culture-dependent approaches (Morovic et al, 2016; Patro et al, 2016)

Methods

Results

Conclusion