Mining for Active Molecules in Probiotic Supernatant by Combining Non-Targeted Metabolomics and Immunoregulation Testing.

Juliano Roldan Fonseca,Marianna Lucio,Mourad Harir,Philippe Schmitt-Kopplin

doi:10.3390/metabo12010035

Abstract

Chronic respiratory diseases such as asthma are highly prevalent in industrialized countries. As cases are expected to rise, there is a growing demand for alternative therapies. Our recent research on the potential benefits of probiotics suggests that they could prevent and reduce the symptoms of many diseases by modulating the host immune system with secreted metabolites. This article presents the first steps of the research that led us to identify the immunoregulatory bioactivity of the amino acid d-Trp reported in our previous study. Here we analyzed the cell culture metabolic footprinting of 25 commercially available probiotic strains to associate metabolic pathway activity information with their respective immune modulatory activity observed in vitro. Crude probiotic supernatant samples were processed in three different ways prior to untargeted analysis in positive and negative ionization mode by direct infusion ESI-FT-ICR-MS: protein precipitation and solid phase extraction (SPE) using HLB and CN-E sorbent cartridges. The data obtained were submitted to multivariate statistical analyses to distinguish supernatant samples into the bioactive and non-bioactive group. Pathway analysis using discriminant molecular features showed an overrepresentation of the tryptophan metabolic pathway for the bioactive supernatant class, suggesting that molecules taking part in that pathway may be involved in the immunomodulatory activity observed in vitro. This work showcases the potential of metabolomics to drive product development and novel bioactive compound discovery out of complex biological samples in a top-down manner.

Highlights

The hygiene hypothesis suggests that the increase of atopic diseases in the modern civilization may be caused by the decreased exposure to microbes during the children’s first year of life, in developed countries and urban areas
Each individual probiotic culture supernatant was harvested at early stationary phase since bacteria are at their maximum size and cell density and, in theory, have max
Prior FT-ICR-MS analysis, each individual probiotic supernatant was subjected to three different desalting extraction procedures to generate three organic extracts using methanol: two after solid phase extraction (HLB-SPE and cyano-propyl sorbent (CN-E) SPE) and one after a simple protein precipitation of crude supernatant

Summary

Introduction

The hygiene hypothesis suggests that the increase of atopic diseases in the modern civilization (e.g., asthma, allergic rhinitis, and atopic dermatitis) may be caused by the decreased exposure to microbes during the children’s first year of life, in developed countries and urban areas. A comparable theory was proposed by Metchnikoff at the beginning of the twentieth century, which is considered to be the precursor of the concept of probiotic bacteria He suggested that the Bulgarian rural population had a longer and healthier life span due to their high consumption of fermented milk containing the bacterium “Bulgarian Bacillus”, today called Lactobacillus bulgaricus [3]. It was only in the mid-1990s that “probiotics” received proper attention from the medical community as potential therapeutic agents [4]. Probiotics represent a serious research field where health claims have been made on a variety of diseases and a multibillion dollar global market has emerged around it, full of popular products that are part of our daily life [5]

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