Development of omics-based protocols for the microbiological characterization of multi-strain formulations marketed as probiotics: the case of VSL#3.

Diego Mora,Stefania Arioli,Sjef Boeren,Rossella Filardi,Steven Aalvink,Willem M De Vos

doi:10.1111/1751-7915.13476

Abstract

SummaryThe growing commercial interest in multi‐strain formulations marketed as probiotics has not been accompanied by an equal increase in the evaluation of quality levels of these biotechnological products. The multi‐strain product VSL#3 was used as a model to setup a microbiological characterization that could be extended to other formulations with high complexity. Shotgun metagenomics by deep Illumina sequencing was applied to DNA isolated from the commercial VSL#3 product to confirm strains identity safety and composition. Single‐cell analysis was used to evaluate the cell viability, and β‐galactosidase and urease activity have been used as marker to monitor the reproducibility of the production process. Similarly, these lots were characterized in detail by a metaproteomics approach for which a robust protein extraction protocol was combined with advanced mass spectrometry. The results identified over 1600 protein groups belonging to all strains present in the VSL#3 formulation. Of interest, only 3.2 % proteins showed significant differences mainly related to small variations in strain abundance. The protocols developed in this study addressed several quality criteria that are relevant for marketed multi‐strain products and these represent the first efforts to define the quality of complex probiotic formulations such as VSL#3.

Highlights

The global sales of cultures marketed as probiotics is increasing every year, and their application is considered instrumental for improving health and well-being
In contrast to starter cultures that are under a strict microbiological quality control because their activity is fundamental for the success of the production process of industrial fermentations, probiotics are currently only controlled at the taxonomic level of the strain(s) used and at the number of viable cells (Tumuola et al, 2001; Patrone et al, 2016; Vecchione et al, 2018)
We developed a series of culture-independent, metagenome and metaproteome-based microbiological characterizations that could be applied for this and other probiotic products to evaluate the species and strain composition, viability and safety, as well as their proteins and some enzymatic activities that could be used to monitor the reproducibility of the biomass production process

Summary

Introduction

The global sales of cultures marketed as probiotics is increasing every year, and their application is considered instrumental for improving health and well-being. This growing scientific and commercial interest has not been accompanied by an equal increase in the evaluation of quality levels for the products that are commercialized (Sanders et al, 2014; Kolace^k et al, 2017). Probiotic core benefits have been described that are shared among commonly used probiotic strains (Hill et al, 2014; Lebeer et al, 2018) Such probiotic benefits comprise operational functions, such as colonization resistance, production of lactic and shortchain fatty acids, regulation of intestinal transit, normalization of perturbed microbiota, increased turnover of enterocytes or competitive exclusion of pathogens. While our fundamental knowledge of the underlying mechanisms is still limited, an increasing number of these core benefits have been linked to specific molecules, structures or metabolic pathways that can be identified or predicted using molecular approaches

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