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Analytical and Bioanalytical Chemistry

, Volume 408, Issue 1, pp 83–96 | Cite as

Metabolic footprinting for investigation of antifungal properties of Lactobacillus paracasei

  • Anders H. HonoréEmail author
  • Stina D. Aunsbjerg
  • Parvaneh Ebrahimi
  • Michael Thorsen
  • Connie Benfeldt
  • Susanne Knøchel
  • Thomas Skov
Paper in Forefront

Abstract

Lactic acid bacteria with antifungal properties are applied for biopreservation of food. In order to further our understanding of their antifungal mechanism, there is an ongoing search for bioactive molecules. With a focus on the metabolites formed, bioassay-guided fractionation and comprehensive screening have identified compounds as antifungal. Although these are active, the compounds have been found in concentrations that are too low to account for the observed antifungal effect. It has been hypothesized that the formation of metabolites and consumption of nutrients during bacterial fermentations form the basis for the antifungal effect, i.e., the composition of the exometabolome. To build a more comprehensive view of the chemical changes induced by bacterial fermentation and the effects on mold growth, a strategy for correlating the exometabolomic profiles with mold growth was applied. The antifungal properties were assessed by measuring mold growth of two Penicillium strains on cell-free ferments of three strains of Lactobacillus paracasei pre-fermented in a chemically defined medium. Exometabolomic profiling was performed by reversed-phase liquid chromatography in combination with mass spectrometry in electrospray positive and negative modes. By multivariate data analysis, the three strains of Lb. paracasei were readily distinguished by the relative difference of their exometabolomes. The relative differences correlated with the relative growth of the two Penicillium strains. Metabolic footprinting proved to be a supplement to bioassay-guided fractionation for investigation of antifungal properties of bacterial ferments. Additionally, three previously identified and three novel antifungal metabolites from Lb. paracasei and their potential precursors were detected and assigned using the strategy.

Keywords

Untargeted profiling LC/MS Multivariate data analysis Minimal inhibitory concentration Antifungal 

Abbreviations

ArAT

Aromatic aminotransferase

BCAA

Branched-chain amino acid

BcAT

Branched-chain aminotransferase

BPC

Base peak chromatogram

CDIM

Chemically defined interaction medium

CF

Cell-free ferment

Da

Dalton

ESI

Electrospray ionization

FID

Flame ionization detector

GC

Gas chromatography

id

Internal diameter

ID

Inhibition degree

IS

Internal standard

LC

Liquid chromatography

m/z

Mass-to-charge ratio

MIC

Minimal inhibitory concentration

MS

Mass spectrometry

Neg

Negative, as for negative electrospray mode

OD

Optical density

PC

Principal component

PCA

Principal component analysis

PLSR

Partial least squares regression

Pos

Positive, as for positive electrospray mode

ppm

Parts per million

REF

Reference, un-inoculated substrate

TIC

Total ion chromatogram

UPLC

Ultra-performance liquid chromatography

VIP

Variable importance in projection

Notes

Acknowledgments

This work was partially financed by the Danish Ministry of Science, Innovation and Higher Education and by the University of Copenhagen as a scholarship for Stina Dissing Aunsbjerg. The analytical support for GC work by Research Associate Marianne Termansen and Technician Lasse Hørup, DuPont Nutritional BioSciences ApS was highly appreciated.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2015_9103_MOESM1_ESM.pdf (995 kb)
ESM 1 (PDF 994 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Anders H. Honoré
    • 1
    • 2
    Email author
  • Stina D. Aunsbjerg
    • 2
  • Parvaneh Ebrahimi
    • 2
  • Michael Thorsen
    • 1
  • Connie Benfeldt
    • 1
  • Susanne Knøchel
    • 2
  • Thomas Skov
    • 2
  1. 1.DuPont Nutrition Biosciences ApSBrabrandDenmark
  2. 2.Department of Food Science, Faculty of ScienceUniversity of CopenhagenFrederiksberg CDenmark

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